Isolated polypeptides of cd44 and uses thereof

ABSTRACT

Isolated polypeptides of CD44 are provided. Accordingly, there is provided an isolated polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3. Also provided is an isolated end-capping modified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, wherein the modified polypeptide comprises an anti-inflammatory activity. Also provided are compositions of matter, fusion proteins and pharmaceutical compositions and their use in the treatment of inflammatory disease.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to anisolated polypeptide of CD44 and, more particularly, but notexclusively, to an isolated polypeptide of CD44vRA and its use in thetreatment of inflammatory disease.

CD44 is a cell surface adhesion molecule involved in multiple cellularfunctions, including cell-cell and cell-matrix interactions, cellmigration, programmed cell death (apoptosis), or, conversely, cellsurvival and proliferation.

CD44 is the major cell surface receptor for hyaluronic acid (HA) but ithas also been shown to bind proteins such as collagens, fibronectin,fibrinogen, laminin, mucosal vascular addressin and osteopontin. CD44 isessential for recruitment of circulating lymphocytes to the site ofinflammation and marked accumulation of CD44, and sometimes hyaluronicacid, is detected in areas of intensive cell migration and cellproliferation, as in wound healing, tissue remodeling, inflammation,morphogenesis and carcinogenesis.

The genomic sequence of mouse and human CD44 includes 5 constant exonsat the 5′ terminus, and 5 constant exons at the 3′ end. The mouse CD44gene includes 10 variant exons in the middle of the molecule, designatedV₁-V₁₀, resulting in a total of 20 exons. The human CD44 gene comprisesonly 9 of these 10 variant exons (V₂-V₁₀) thus comprising a total of 19exons. Differential V₂-V₁₀ alternative splicing generates many isoformsof CD44 that express various combinations of variant exons (designatedexon Vx, x=1-10), which are inserted in the membrane proximal domain andconstitute the variable region of the molecule. These molecules aredesignated CD44 variants (CD44v). A few dozens isoforms of CD44 areknown to date.

CD44s, which does not contain any variant exon, is the most ubiquitousform and is expressed by most cell types [Ponta, H., et al. Nat Rev MolCell Biol. 2003 January; 4(1):33-45]. CD44 variant proteins, in whichone or more of the 10 variant exons are included, are mostly reported inassociation with cancer, and autoimmune diseases such as rheumatoidarthritis and multiple sclerosis [see e.g. Naor et al. Adv. Cancer Res.,71, 241-319, 1997; and Naor et al. Critical Reviews in ClinicalLaboratory Sciences. 39, 527-579, 2002].

Joint inflammatory cells of patients with rheumatoid arthritis (RA)display a sequence of alternatively spliced CD44 variant designatedCD44vRA. Human CD44vRA contains the same sequence as that ofkeratinocytes CD44v3-v10 isoform with an addition of extra alanine inthe splicing junction between variant exon 4 and variant exon 5, whichdoes not interfere with the reading frame. Mice with collagen-inducedarthritis (CIA) contain at the same site a similar sequence that alsoincludes the alanine. The CD44vRA sequence is expressed on jointinflammatory synovial cells of RA patients and Psoriatic Arthritis (PA)patients, but neither on keratinocytes nor peripheral blood leukocytes(PBLs) of healthy donors. Furthermore, while joint inflammatory cells ofRA patients express CD44vRA, PBLs from the same patients and synovialfluid cells from osteoarthritis patients hardly express this variant,demonstrating the exclusivity of this isoform. (Nedvetzki et al., J ClinInvest 111:1211-1220, 2003; Golan et al., J Autoimm 28:99-113, 2007).

It has been reported that administration of anti-CD44 antibodies, CD44proteins, peptides or derivatives can be used for treating variousautoimmune diseases (e.g. Naor et al., Adv. Cancer Res., 71, 241-319,1997; Naor et al., Critical Reviews in Clinical Laboratory Sciences. 39,527-579, 2002; Turley E A, and Naor D. Front Biosci. 17:1775-1794,2012). In addition, anti-CD44vRA monoclonal antibodies andCD44vRA-derived peptides were previously suggested (Golan et al., JAutoimm 28:99-113, 2007, International Application Publication Nos:WO2010/058396, WO 2005/007700; WO 2003/014160, WO 2000/075312; U.S. Pat.Nos. 7,534,605 and 8,193,311; and US Patent Application Publication No:US 20060019340).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated polypeptide consisting of an amino acidsequence selected form the group consisting of SEQ ID NOs: 1-3.

According to an aspect of some embodiments of the present inventionthere is provided an isolated end-capping modified polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1-3, wherein the modified polypeptide comprises ananti-inflammatory activity.

According to some embodiments of the invention, the end-cappingcomprises an N terminus end-capping.

According to some embodiments of the invention, the N terminusend-capping comprises an Acetyl.

According to some embodiments of the invention, the end-cappingcomprises a C terminus end-capping.

According to some embodiments of the invention, the C terminusend-capping comprises an Amide.

According to some embodiments of the invention, the polypeptide consistsof an amino acid sequence selected form the group consisting of SEQ IDNOs: 1-3.

According to some embodiments of the invention, the polypeptide is asset forth in SEQ ID NO: 1.

According to some embodiments of the invention, the end-capping modifiedpolypeptide being selected form the group consisting to SEQ ID NOs: 4-6.

According to some embodiments of the invention there is provided acomposition of matter comprising the isolated polypeptide and anon-proteinaceous moiety attached to the isolated polypeptide, whereinthe isolated fusion polypeptide comprises an anti-inflammatory activity.

According to some embodiments of the invention there is provided anisolated fusion polypeptide comprising the isolate polypeptide having aC and/or N terminally attached amino acid sequence, wherein the Cterminally amino acid sequence is a non-contiguous CD44vRA amino acidsequence with the isolated fusion polypeptide; and wherein the fusionpolypeptide comprises an anti-inflammatory activity.

According to some embodiments of the invention, the attached is covalentattachment.

According to some embodiments of the invention, the anti-inflammatoryactivity is not dependent on vaccination or mucosal tolerance.

According to some embodiments of the invention, the isolated polypeptideor the composition of matter being capable of binding a protein selectedfrom the group consisting of serum amyloid A, Transthyretin andapolipoprotein B.

According to some embodiments of the invention there is provided apharmaceutical composition comprising as an active agent the isolatedpolypeptide or the composition of matter; and a pharmaceuticallyacceptable carrier or diluent.

According to some embodiments of the invention there is provided amethod of treating an inflammatory disease in a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of the isolated polypeptide, the composition of matteror the pharmaceutical composition, thereby treating the inflammatorydisease in the subject.

According to some embodiments of the invention there is provided a useof the isolated polypeptide, the composition of matter or thepharmaceutical composition, for the manufacture of a medicament for thetreatment of an inflammatory disease.

According to some embodiments of the invention, the administeringcomprises oral administering.

According to some embodiments of the invention, the composition isformulated for oral administration.

According to some embodiments of the invention, the inflammatory diseaseinvolves cells expressing CD44vRA.

According to some embodiments of the invention, the inflammatory diseaseis selected from the group consisting of Rheumatoid arthritis, psoriaticarthritis, Alzheimer's disease, cancer and cardiovascular disease.

According to some embodiments of the invention, the inflammatory diseaseis Rheumatoid arthritis.

According to some embodiments of the invention, there is provided anisolated polynucleotide comprising a nucleic acid sequence encoding theisolated polypeptide.

According to some embodiments of the invention there is provided anucleic acid construct comprising the isolated polynucleotide.

According to some embodiments of the invention there is provided amethod of determining potency of a batch of the isolated polypeptide,the composition of matter or the pharmaceutical composition, the methodcomprising:

(a) contacting a batch of the isolated polypeptide, the composition ofmatter or the pharmaceutical composition with fibroblasts obtained froman inflammatory joint of a Rheumatoid arthritis patient; and

(b) determining survival of the fibroblasts following a predeterminedincubation time, so as to determine the potency of the batch.

According to some embodiments of the invention, the method comprisingsynthesizing the isolated polypeptide, the composition of matter or thepharmaceutical composition with a modification prior to the contacting.

According to some embodiments of the invention, reduced survival of thefibroblasts following the contacting is indicative that the batch ispotent.

According to some embodiments of the invention, the method comprisingcomparing the survival of the cells with survival of the cells followingcontacting with a reference standard batch of the isolated polypeptide,the composition of matter or the pharmaceutical composition, so as todetermine the relative potency of the batch.

According to some embodiments of the invention, the method is effectedin-vitro or ex-vivo.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 shows Liquid chromatography-mass spectrometry (LCMS) analysisdemonstrating the % stability of the 5-mer RA peptide (SEQ ID NO: 1)following storage at the indicated temperatures; assuming that storageat −20° C. represents 100% stability.

FIGS. 2A-C are graphs demonstrating that the 9-mer RA peptide (SEQ IDNO: 3) reduces joint inflammation in collagen-induced arthritis (CIA)mice on DBA/1 background. The Figures show paw swelling followinginjection of the 9-mer peptide at a dose of 25 μg (FIG. 2A), 100 μg(FIG. 2B) or 150 μg (FIG. 2C) at the indicated time points (marked byarrow heads). PBS was injected as control. The y-axis represents Δ pawswelling indicating the difference (by mm) between the width of the pawat each of the measurement time points and the width of the paw at theonset of disease (time 0). The results are expressed as mean±SE; thenumber of mice in each group (n) is indicated in insets of eachFigure; * P<0.05.

FIGS. 3A-C demonstrate that treatment with the 5-mer RA peptide (SEQ IDNO: 1) can restore normal histology of the inflamed joint in CIA mice onC57BL/6 background. FIGS. 3A and 3B are representative photomicrographsof H&E stained hind limb joint sections from mice treated with PBScontrol (FIG. 3A) or 5-mer RA peptide (FIG. 3B). FIG. 3C is a graphsummarizing the average inflammatory score as evaluated by histologicalexamination of H&E stained hind limb joint sections from mice treatedwith PBS control (n=7) or 5-mer RA peptide (n=7), wherein 0 indicates noinfiltration and 4 indicates massive infiltration. p<0.0001.

FIG. 4 is a graph demonstrating that the 7- and 9-mer protected RApeptides (SEQ ID NOs: 5-6) reduce joint inflammation in CIA mice onDBA/1 background. The Figure shows paw swelling following injection ofthe peptides at a dose of 200 μg at the indicated time points (marked byarrows). PBS was injected as control. The y-axis represents Δ pawswelling indicating the difference (by mm) between the width of the pawat each of the measurement time points and the width of the paw at theonset of disease (time 0). The results are expressed as mean±SE; thenumber of mice in each group (n) is indicated in insets of eachFigure; * P<0.05, ** p<0.01.

FIGS. 5A-B are graphs demonstrating that the 5-mer protected RA peptide(SEQ ID NO: 4) reduces joint inflammation in CIA mice on DBA/1background. The Figures show paw swelling following injection of thepeptide at a dose of 200 μg at the indicated time points (marked byarrows). PBS (FIGS. 5A and 5B) or Dexamethasone (Dex) (FIG. 5A) wereinjected as control. The y-axis represents Δ paw swelling indicating thedifference (by mm) between the width of the paw at each of themeasurement time points and the width of the paw at the onset of disease(time 0). The results are expressed as mean±SE; the number of mice ineach group (n) is indicated in insets of each Figure; * P<0.05, **p<0.01.

FIG. 6 is a graph demonstrating that the 5-mer protected RA peptide (SEQID NO: 4) reduces joint inflammation in CIA mice on C57BL background.The Figures show paw swelling following injection of the peptide at adose of 70 μg for 10 consecutive days following onset of disease. PBSwas injected as control. The y-axis represents Δ paw swelling indicatingthe difference (by mm) between the width of the paw at each of themeasurement time points and the width of the paw at the onset of disease(time 0). The results are expressed as mean±SE; * P<0.006.

FIGS. 7A-C are graphs demonstrating that the 7-mer protected RA peptide,which includes the core MTADV sequence (SEQ ID NO: 5), reduces jointinflammation in CIA mice on DBA/1 background, while the non-specificcore scrambled 7-mer peptide (SEQ ID NO: 7) has no effect on jointinflammation in this model. The Figures show paw swelling followinginjection of the peptides at a dose of 200 μg at the indicated timepoints (marked by arrows). PBS was injected as control. The y-axisrepresents paw swelling in mm (FIGS. 7A and 7C) or Δ paw swelling (FIG.7B) indicating the difference between the width of the paw at each ofthe measurement time points and the width of the paw at the onset ofdisease (time 0, FIG. 7B). The results are expressed as mean±SE; thenumber of mice in each group (n) is indicated in insets of eachFigure; * P<0.05, ** p<0.005.

FIG. 8 is a bar graph showing the percentages of healthy hind paws inCIA mice following injection of 7-mer protected RA peptide (SEQ ID NO:5) or non-specific scrambled 7-mer peptide (SEQ ID NO: 7) according tothe experimental method described in Table 6. PBS was injected ascontrol.

FIGS. 9A-B are graphs demonstrating that a dose of 70 μg per injectionis the optimal dose for inhibiting joint inflammation in CIA mice onC57BL/6 background by the 5-mer protected RA peptide (SEQ ID NO: 4). TheFigures show paw swelling following injection of the peptide at a doseof 70, 200 and 600 μg (FIG. 9A) or 10, 25 and 70 μg (FIG. 9B) for 10consecutive days following onset of disease. PBS was injected ascontrol. The y-axis represents Δ paw swelling indicating the differencebetween the width of the paw at each of the measurement time points andthe width of the paw at the onset of disease (time 0). The results areexpressed as mean±SE; the number of mice in each group (n) is indicatedin insets of each graph; * P is indicated on each graph.

FIG. 10 shows graphs demonstrating the effect of the 5-mer RA peptide(SEQ ID NO: 1) on delayed type hypersensitivity (DTH) response inC57BL/6 mice. The y-axis represents the difference in thickness betweenthe right and the left ears on day 7. Treatment with PBS and anti-TNFαserved as positive and negative control, respectively. The results areexpressed as mean±SE. The DTH protocol comprised sensitization withOxazolone on day 0; elicitation (challenge) in the ear with Oxazolone onday 6; and measurement of ear thickness day 7. PBS or peptide wereinjected from day −1 to day 7.

FIG. 11 is a graph showing absence of neutralizing anti-peptide specificantibodies in the serum of mice treated with the 5-mer peptide (SEQ IDNO: 1), as determined by ELISA. ELISA plates coated with the 5-merpeptide or with collagen and mouse IgG, which served as positivecontrols. Sera from mice treated with the 5-mer peptide or with PBS wereadded to plate wells. Serum from naïve mice and mice treated with PBSserved as negative controls.

FIG. 12 is a schematic representation of the procedure used foridentification of the 5-mer peptide target proteins.

FIG. 13 is a graph showing the pharmacokinetic elimination of the 5-merRA peptide (SEQ ID NO: 1) in the serum of mice following a singleinjection of the peptide.

FIG. 14 is a graph demonstrating the in vitro effect of the 5-mer RApeptide (SEQ ID NO: 1) on survival of fibroblasts isolated from theinflammatory joint of an RA patient, as determined by a MTT assay.

FIG. 15 is a graph demonstrating that Serum Amyloid A (SAA) prevents thein vitro effect of the 5-mer RA peptide (SEQ ID NO: 1) on survival offibroblasts isolated from the inflammatory joint of an RA patient, asdetermined by a MTT assay. Lactalbumin (LA) was used as a non-specificcontrol. The 5-mer peptide was added in a constant concentration (25μg/ml); the x-axis indicates SAA and LA concentration.

FIG. 16 is a graph demonstrating the in vitro effect of the 5-mer RApeptide (SEQ ID NO: 1) on survival of fibroblasts isolated from theinflammatory joint of an RA patient in comparison to the 5-mer protectedRA peptide (SEQ ID NO: 4), as determined by a MTT assay.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to anisolated polypeptide of CD44 and, more particularly, but notexclusively, to an isolated polypeptide of CD44vRA and its use in thetreatment of inflammatory disease.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

CD44 is a cell surface adhesion molecule involved in multiple cellularfunctions, including cell-cell and cell-matrix interactions, cellmigration, programmed cell death or, conversely, cell survival andproliferation. The genomic sequence of human CD44 includes 5 constantexons at the 5′ terminus and 5 constant exons at the 3′ terminus, aswell as 9 variant exons encompassed therebetween. Several dozens ofsplice variants of CD44 are known to date. CD44s (SEQ ID NO: 9), whichdoes not contain any variant exon, is the most ubiquitous form and isexpressed by most cell types. Joint inflammatory cells of patients withpsoriatic arthritis (PA), rheumatoid arthritis (RA) present a sequenceof alternatively spliced CD44 variant designated CD44vRA (SEQ ID NO:11), not expressed on keratinocytes nor peripheral blood leukocytes(PBLs) of healthy donors.

Whilst reducing the present invention to practice, the present inventorshave now uncovered that peptides as short as 5, 7 or 9 mers comprising aMTADV sequence resulting from inclusion of alanine in the splicingjunction between variant exon 4 and variant exon 5 of CD44vRA arecapable of inhibiting joint inflammation in a CIA mouse model (the mouseanalogue of human RA). Without wishing to be bound by theory it isbelieved that the polypeptides of some embodiments of the inventionelicit their activity by competing with the natural ligand of CD44vRA.

As is illustrated hereinunder and in the examples section, whichfollows, the present inventors have synthesized 5-, 7- and 9-merpeptides (SEQ ID NOs: 1-3, also denoted herein as “RA peptides”) andrespective peptides with Acetyl and Amide protecting residues, at theamino and carboxyl terminal ends of the peptides respectively (SEQ IDNOs: 4-6, also denoted herein as “RA protected peptides”). The peptidescomprise hydrophobic amino acids, no proteolytic sites and are stable atroom temperature and 4° C. for at least 22 weeks (Example 1, FIG. 1).The synthesized RA peptides and RA protected peptides were able toreduce joint inflammation in-vivo in a CIA mouse model (Examples 2-4,FIGS. 2A-B, 3A-C; 4, 5-A-B, 6, 8 and 9A-B). Moreover, the peptides didnot elicit generation of neutralizing anti-peptide specific antibodiesnor affected general immune response as evaluated by delayedhypersensitivity (DTH) response (Example 5-6, FIGS. 10-11). Importantly,a scrambled non-specific 7-mer protected peptide (SEQ ID NO: 7) had noeffect on joint inflammation in the CIA mouse model (Example 3 FIGS.7A-B and 8). Mass spectrometry analysis further revealed few potentialtarget proteins of the RA peptides, namely Serum amyloid A,Transthyretin and Apolipoprotein B (Example 7, FIG. 12). In addition theinventors have developed a novel in-vitro assay to test the activity ofthe RA peptides and RA protected peptides (Example 9, FIGS. 14-16).

Consequently, the present teachings suggest the use of compositionscomprising the RA- and RA-protected peptides in the treatment ofinflammatory diseases.

Thus, according to a first aspect of the present invention, there isprovided an isolated polypeptide consisting of an amino acid sequenceselected form the group consisting of SEQ ID NOs: 1-3.

According to specific embodiments the polypeptide is as set forth in SEQID NO: 1.

According to specific embodiments the polypeptide is as set forth in SEQID NO: 2.

According to specific embodiments the polypeptide is as set forth in SEQID NO: 3.

According to an aspect of the present invention, there is provided anisolated end-capping modified polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1-3, whereinsaid modified polypeptide comprises an anti-inflammatory activity.

According to specific embodiments, the polypeptide amino acid sequenceof the end-capping modified polypeptide consists of an amino acidsequence selected form the group consisting of SEQ ID NOs: 1-3.

According to another aspect of the present invention there is provided acomposition of matter comprising the isolated polypeptide and anon-proteinaceous moiety attached to the isolated polypeptide, whereinthe isolated fusion polypeptide comprises an anti-inflammatory activity.

According to another aspect of the present invention there is providedan isolated fusion polypeptide comprising the isolated polypeptidehaving a C and/or N terminally attached amino acid sequence, whereinsaid C terminally amino acid sequence is a non-contiguous CD44vRA aminoacid sequence with said isolated fusion polypeptide; and wherein saidfusion polypeptide comprises an anti-inflammatory activity.

The terms “peptide” and “polypeptide” which are interchangeably usedherein encompass native peptides (either degradation products,synthetically synthesized peptides or recombinant peptides) andpeptidomimetics (typically, synthetically synthesized peptides), as wellas peptoids and semipeptoids which are peptide analogs, which may have,for example, modifications rendering the peptides more stable while in abody, more capable of penetrating into cells improving clearance,biodistribution and/or pharmacokinetics. Such modifications include, butare not limited to N terminus modification, C terminus modification,peptide bond modification, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds(—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds(—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g.,methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds(—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic doublebonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presenton the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (e.g. 2-3) bonds at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bynon-natural aromatic amino acids such as1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine,ring-methylated derivatives of Phe, halogenated derivatives of Phe orO-methyl-Tyr.

The peptides of some embodiments of the invention may also include oneor more modified amino acids or one or more non-amino acid monomers(e.g. fatty acids, complex carbohydrates etc).

The term “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodemosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids (stereoisomers).

Tables 1 and 2 below list naturally occurring amino acids (Table 1), andnon-conventional or modified amino acids (e.g., synthetic, Table 2)which can be used with some embodiments of the invention.

TABLE 1 Three-Letter One-letter Amino Acid Abbreviation Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE 2 Non-conventional Non-conventional amino acid Code amino acidCode ornithine Orn hydroxyproline Hyp α-aminobutyric acid Abuaminonorbornyl- Norb carboxylate D-alanine Dala aminocyclopropane- Cprocarboxylate D-arginine Darg N-(3- Narg guanidinopropyl)glycineD-asparagine Dasn N- Nasn (carbamylmethyl)glycine D-aspartic acid DaspN-(carboxymethyl)glycine Nasp D-cysteine Dcys N-(thiomethyl)glycine NcysD-glutamine Dgln N-(2- Ngln carbamylethyl)glycine D-glutamic acid DgluN-(2-carboxyethyl)glycine Nglu D-histidine DhisN-(imidazolylethyl)glycine Nhis D-isoleucine DileN-(1-methylpropyl)glycine Nile D-leucine Dleu N-(2-methylpropyl)glycineNleu D-lysine Dlys N-(4-aminobutyl)glycine Nlys D-methionine Dmet N-(2-Nmet methylthioethyl)glycine D-ornithine Dorn N-(3-aminopropyl)glycineNorn D-phenylalanine Dphe N-benzylglycine Nphe D-proline DproN-(hydroxymethyl)glycine Nser D-serine Dser N-(1-hydroxyethyl)glycineNthr D-threonine Dthr N-(3-indolylethyl) glycine Nhtrp D-tryptophan DtrpN- Ntyr (p-hydroxyphenyeglycine D-tyrosine Dtyr N-(1-methylethyl)glycineNval D-valine Dval N-methylglycine Nmgly D-N-methylalanine DnmalaL-N-methylalanine Nmala D-N-methylarginine Dnmarg L-N-methylarginineNmarg D-N-methylasparagine Dnmasn L-N-methylasparagine NmasnD-N-methylasparatate Dnmasp L-N-methylaspartic acid NmaspD-N-methylcysteine Dnmcys L-N-methylcysteine Nmcys D-N-methylglutamineDnmgln L-N-methylglutamine Nmgln D-N-methylglutamate DnmgluL-N-methylglutamic acid Nmglu D-N-methylhistidine DnmhisL-N-methylhistidine Nmhis D-N-methylisoleucine DnmileL-N-methylisolleucine Nmile D-N-methylleucine Dnmleu L-N-methylleucineNmleu D-N-methyllysine Dnmlys L-N-methyllysine NmlysD-N-methylmethionine Dnmmet L-N-methylmethionine NmmetD-N-methylornithine Dnmorn L-N-methylornithine Nmorn D-N- DnmpheL-N-methylphenylalanine Nmphe methylphenylalanine D-N-methylprolineDnmpro L-N-methylproline Nmpro D-N-methylserine Dnmser L-N-methylserineNmser D-N-methylthreonine Dnmthr L-N-methylthreonine NmthrD-N-methyltryptophan Dnmtrp L-N-methyltryptophan NmtrpD-N-methyltyrosine Dnmtyr L-N-methyltyrosine Nmtyr D-N-methylvalineDnmval L-N-methylvaline Nmval L-norleucine Nle L-N-methylnorleucineNmnle L-norvaline Nva L-N-methylnorvaline Nmnva L-ethylglycine EtgL-N-methyl-ethylglycine Nmetg L-t-butylglycine Tbug L-N-methyl-t- Nmtbugbutylglycine L-homophenylalanine Hphe L-N-methyl- Nmhphehomophenylalanine α-naphthylalanine Anap N-methyl-α- Nmanapnaphthylalanine penicillamine Pen N-methylpenicillamine Nmpenγ-aminobutyric acid Gabu N-methyl-γ- Nmgabu aminobutyratecyclohexylalanine Chexa N-methyl- Nmchexa cyclohexylalaninecyclopentylalanine Cpen N-methyl- Nmcpen cyclopentylalanine α-amino-α-Aabu N-methyl-α-amino-α- Nmaabu methylbutyrate methylbutyrateα-aminoisobutyric acid Aib N-methyl-α- Nmaib aminoisobutyrateD-α-methylarginine Dmarg L-α-methylarginine Marg D-α-methylasparagineDmasn L-α-methylasparagine Masn D-α-methylaspartate DmaspL-α-methylaspartate Masp D-α-methylcysteine Dmcys L-α-methylcysteineMcys D-α-methylglutamine Dmgln L-α-methylglutamine Mgln D-α-methyl DmgluL-α-methylglutamate Mglu glutamic acid D-α-methylhistidine DmhisL-α-methylhistidine Mhis D-α-methylisoleucine Dmile L-α-methylisoleucineMile D-α-methylleucine Dmleu L-α-methylleucine Mleu D-α-methyllysineDmlys L-α-methyllysine Mlys D-α-methylmethionine DmmetL-α-methylmethionine Mmet D-α-methylornithine Dmorn L-α-methylornithineMorn D-α- Dmphe L-α-methylphenylalanine Mphe methylphenylalanineD-α-methylproline Dmpro L-α-methylproline Mpro D-α-methylserine DmserL-α-methylserine Mser D-α-methylthreonine Dmthr L-α-methylthreonine MthrD-α-methyltryptophan Dmtrp L-α-methyltryptophan Mtrp D-α-methyltyrosineDmtyr L-α-methyltyrosine Mtyr D-α-methylvaline Dmval L-α-methylvalineMval N-cyclobutylglycine Ncbut L-α-methylnorvaline MnvaN-cycloheptylglycine Nchep L-α-methylethylglycine MetgN-cyclohexylglycine Nchex L-α-methyl-t-butylglycine MtbugN-cyclodecylglycine Ncdec L-α-methyl- Mhphe homophenylalanineN-cyclododecylglycine Ncdod α-methyl-α- Manap naphthylalanineN-cyclooctylglycine Ncoct α-methylpenicillamine MpenN-cyclopropylglycine Ncpro α-methyl-γ-aminobutyrate MgabuN-cycloundecylglycine Ncund α-methyl- Mchexa cyclohexylalanineN-(2-aminoethyl)glycine Naeg α-methyl- Mcpen cyclopentylalanine N-(2,2-Nbhm N-(N-(2,2-diphenylethyl) Nnbhm diphenylethyl)glycinecarbamylmethyl-glycine N-(3,3- Nbhe N-(N-(3,3-diphenylpropyl) Nnbhediphenylpropyl)glycine carbamylmethyl-glycine 1-carboxy-1-(2,2- Nmbc1,2,3,4- Tic diphenyl tetrahydroisoquinoline- ethylamino)cyclopropane3-carboxylic acid phosphoserine pSer phosphothreonine pThrphosphotyrosine pTyr O-methyl-tyrosine 2-aminoadipic acid hydroxylysine

The amino acids of the polypeptides of the present invention may besubstituted either conservatively or non-conservatively.

The term “conservative substitution” as used herein, refers to thereplacement of an amino acid present in the native sequence in thepeptide with a naturally or non-naturally occurring amino or apeptidomimetics having similar steric properties. Where the side-chainof the native amino acid to be replaced is either polar or hydrophobic,the conservative substitution should be with a naturally occurring aminoacid, a non-naturally occurring amino acid or with a peptidomimeticmoiety which is also polar or hydrophobic (in addition to having thesame steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according totheir properties, conservative substitutions by naturally occurringamino acids can be easily determined bearing in mind the fact that inaccordance with the invention replacement of charged amino acids bysterically similar non-charged amino acids are considered asconservative substitutions.

For producing conservative substitutions by non-naturally occurringamino acids it is also possible to use amino acid analogs (syntheticamino acids) well known in the art. A peptidomimetic of the naturallyoccurring amino acid is well documented in the literature known to theskilled practitioner.

When affecting conservative substitutions the substituting amino acidshould have the same or a similar functional group in the side chain asthe original amino acid.

The phrase “non-conservative substitutions” as used herein refers toreplacement of the amino acid as present in the parent sequence byanother naturally or non-naturally occurring amino acid, havingdifferent electrochemical and/or steric properties. Thus, the side chainof the substituting amino acid can be significantly larger (or smaller)than the side chain of the native amino acid being substituted and/orcan have functional groups with significantly different electronicproperties than the amino acid being substituted. Examples ofnon-conservative substitutions of this type include the substitution ofphenylalanine or cycohexylmethyl glycine for alanine, isoleucine forglycine, or —NH—CH[(—CH₂)₅—COOH]—CO— for aspartic acid. Thosenon-conservative substitutions which fall under the scope of the presentinvention are those which still constitute a peptide havingneuroprotective properties.

The peptides of some embodiments of the invention are preferablyutilized in a linear form, although it will be appreciated that in caseswhere cyclicization does not severely interfere with peptidecharacteristics, cyclic forms of the peptide can also be utilized.

Since the present peptides are preferably utilized in therapeutics whichrequires the peptides to be in soluble form, the peptides of someembodiments of the invention preferably include one or more non-naturalor natural polar amino acids, including but not limited to serine andthreonine which are capable of increasing peptide solubility due totheir hydroxyl-containing side chain.

As mentioned, the N and C termini of the peptides of the presentinvention may be protected by functional groups. Suitable functionalgroups are described in Green and Wuts, “Protecting Groups in OrganicSynthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachingsof which are incorporated herein by reference. Thus, the polypeptide maybe modified at the N-(amine) terminus and/or the C-(carboxyl) terminusthereof so as to produce an end capping modified peptide.

As used herein, the phrases “end-capping modified polypeptide” and“protected polypeptide”, which are interchangeably used herein, refer toa polypeptide which has been modified at the N-(amine) terminus and/orthe C-(carboxyl) terminus thereof. The end-capping modification refersto the attachment of a chemical moiety to the terminus of thepolypeptide, so as to form a cap. Such a chemical moiety is referred toherein as an end capping moiety and is typically also referred to hereinand in the art, interchangeably, as a peptide protecting moiety orgroup. Hydroxyl protecting groups include but are not limited to esters,carbonates and carbamate protecting groups. Amine protecting groupsinclude but are not limited to alkoxy and aryloxy carbonyl groups.Carboxylic acid protecting groups include but are not limited toaliphatic, benzylic and aryl esters.

The phrase “end-capping moiety”, as used herein, refers to a moiety thatwhen attached to the terminus of the peptide, modifies the N and/or Cterminal ends(s) of the peptide. The end-capping modification typicallyresults in masking the charge of the peptide terminus, and/or alteringchemical features thereof, such as, hydrophobicity, hydrophilicity,reactivity, solubility and the like. By selecting the nature of the endcapping modification, the hydrophobicity/hydrophilicity, as well as thesolubility of the peptide can be finely controlled. According tospecific embodiments, the protecting groups facilitate transport of thepeptide attached thereto into a cell. These moieties can be cleaved invivo, either by hydrolysis or enzymatically, inside the cell.

According to specific embodiments, the end-capping modification does notcompromise the biological activity (i.e. anti-inflammatory activity) ofthe polypeptide. Examples of moieties suitable for peptide end-cappingmodification can be found, for example, in Green et al., “ProtectiveGroups in Organic Chemistry”, (Wiley, 2.sup.nd ed. 1991) and Harrison etal., “Compendium of Synthetic Organic Methods”, Vols. 1-8 (John Wileyand Sons, 1971-1996).

According to specific embodiments, the end-capping comprises an Nterminus end-capping.

Representative examples of N-terminus end-capping moieties include, butare not limited to, formyl, acetyl (also denoted herein as “Ac”),trifluoroacetyl, benzyl, benzyloxycarbonyl (also denoted herein as“Cbz”), tert-butoxycarbonyl (also denote d herein as “Boc”),trimethylsilyl (also denoted “TMS”), 2-trimethylsilyl-ethanesulfonyl(also denoted “SES”), trityl and substituted trityl groups,allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (also denoted herein as“Fmoc”), and nitro-veratryloxycarbonyl (“NVOC”).

According to specific embodiments, the N terminus end-capping comprisesan Acetyl.

According to specific embodiments, the end-capping comprises a Cterminus end-capping.

Representative examples of C-terminus end-capping moieties are typicallymoieties that lead to acylation of the carboxy group at the C-terminusand include, but are not limited to, benzyl and trityl ethers as well asalkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, allylethers, monomethoxytrityl and dimethoxytrityl. Alternatively the —COOHgroup of the C-terminus end-capping may be modified to an amide group.

According to specific embodiments, the C terminus end-capping comprisesan Amide.

Other end-capping modifications of peptides include replacement of theamine and/or carboxyl with a different moiety, such as hydroxyl, thiol,halide, alkyl, aryl, alkoxy, aryloxy and the like.

According to specific embodiments, the peptide is modified only at theN-terminus or the C-terminus thereof.

According to other specific embodiments, the peptide is modified at boththe N-terminus and the C-terminus.

According to specific embodiments, the peptide is modified at theN-terminus with an Acetyl and at the C terminus with an Amide.

According to specific embodiments the end-capping modified polypeptideis selected form the group consisting to SEQ ID NOs: 4-6.

The present invention further provides polypeptide conjugates and fusionpolypeptides comprising peptides, analogs and derivatives according tothe invention.

Thus, as mentioned, according to an aspect of the present inventionthere is provided an isolated fusion polypeptide comprising the isolatedpolypeptide having a C and/or N terminally attached amino acid sequence,wherein said C terminally amino acid sequence is a non-contiguousCD44vRA amino acid sequence with said isolated fusion polypeptide; andwherein said fusion polypeptide comprises an anti-inflammatory activity.

As used herein, the phrase “non-contiguous CD44vRA amino acid sequence”refers to a fusion polypeptide that does not comprise an amino acidsequence of SEQ ID NOs: 1, 2 or 3 directly attached in its C terminus toan amino acid sequence of CD44vRA starting at coordinates 306, 308 or308, respectively, of SEQ ID NO: 11.

According to specific embodiments, the isolated polypeptide and theattached amino acid sequence are covalently attached, directly orthrough a spacer or a linker which can be a synthetic or an amino acidlinker.

As used herein the term “CD44” refers to the cell surface protein thatis expressed in a large number of mammalian cell types and is encoded bythe CD44 gene. According to a specific embodiment the CD44 is the humanCD44 gene. The standard isoform, designated CD44, comprising exons 1-5and 16-20 is expressed in most cell types and is set forth in GeneBankAccession Numbers NM_000610 and NP_000601 (SEQ ID NOs: 8 and 9).

As used herein the term “CD44vRA” (SEQ ID NO: 10 and 11) refers to aCD44 variant which is expressed in inflammation sites, e.g. on synovialfluid cells of RA patients but not on PBLs of healthy individuals.CD44vRA variant is a naturally occurring sequence which is presumablyproduced by alternative splicing of the primary transcript of the knownCD44 gene which occurs in cells in inflammatory sites (e.g. in joints ofRA patients) and does not arise from truncation or mutation of the knownCD44 gene. This CD44vRA variant sequence comprises Exons 1-5, 15-17 and19 of the constant part of the CD44 gene as well as Exons 7-14 (v3-v10)of the variable region of the gene. The variant coding sequencecomprises three additional bases (CAG) that are transcribed from the endof the intron bridging Exon v4 to Exon v5 and are inserted at the 5′ endof Exon v5. This extra CAG sequence results in the insertion of a newcodon for the amino acid alanine in position 303 of SEQ ID NO: 11 whileleaving the reading frame intact.

The terms “CD44” and “CD44vRA”, also refer to CD44 and CD44vRAhomologues which exhibit the desired activity (e.g. cell migrationand/or cell-cell and cell-matrix interactions). Such homologues can be,for example, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical or homologous to the polypeptide of SEQ IDNOs: 9 and 11, or 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical to the polynucleotide sequence encodingsame.

The homolog may also refer to an ortholog, a deletion, insertion, orsubstitution variant, including an amino acid substitution, as long asit retains the activity.

Sequence identity or homology can be determined using any protein ornucleic acid sequence alignment algorithm such as Blast, ClustalW, andMUSCLE.

According to other specific embodiments of the invention, the peptide isattached to a non-proteinaceous moiety.

According to specific embodiments, the isolated polypeptide and theattached non-proteinaceous moiety are covalently attached, directly orthrough a spacer or a linker.

The phrase “non-proteinaceous moiety” as used herein refers to amolecule not including peptide bonded amino acids that is attached tothe above-described peptide. According to a specific embodiment thenon-proteinaceous is a non-toxic moiety. Exemplary non-proteinaceousmoieties which may be used according to the present teachings include,but are not limited to a drug, a chemical, a small molecule, apolynucleotide, a detectable moiety, polyethylene glycol (PEG),Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), anddivinyl ether and maleic anhydride copolymer (DIVEMA). According tospecific embodiments of the invention, the non-proteinaceous moietycomprises polyethylene glycol (PEG).

Such a molecule is highly stable (resistant to in-vivo proteolyticactivity probably due to steric hindrance conferred by thenon-proteinaceous moiety) and may be produced using common solid phasesynthesis methods which are inexpensive and highly efficient, as furtherdescribed hereinbelow. However, it will be appreciated that recombinanttechniques may still be used, whereby the recombinant peptide product issubjected to in-vitro modification (e.g., PEGylation as furtherdescribed hereinbelow).

Bioconjugation of the polypeptide amino acid sequence with PEG (i.e.,PEGylation) can be effected using PEG derivatives such asN-hydroxysuccinimide (NHS) esters of PEG carboxylic acids,monomethoxyPEG₂-NHS, succinimidyl ester of carboxymethylated PEG(SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethersof PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC),PEG aldehydes, PEG-orthopyridyl-disulfide, carbonyldimidazol-activatedPEGs, PEG-thiol, PEG-maleimide. Such PEG derivatives are commerciallyavailable at various molecular weights [See, e.g., Catalog, PolyethyleneGlycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle,Ala.)]. If desired, many of the above derivatives are available in amonofunctional monomethoxyPEG (mPEG) form. In general, the PEG added tothe polypeptide of the present invention should range from a molecularweight (MW) of several hundred Daltons to about 100 kDa (e.g., between3-30 kDa). Larger MW PEG may be used, but may result in some loss ofyield of PEGylated polypeptides. The purity of larger PEG moleculesshould be also watched, as it may be difficult to obtain larger MW PEGof purity as high as that obtainable for lower MW PEG. It is preferableto use PEG of at least 85% purity, and more preferably of at least 90%purity, 95% purity, or higher. PEGylation of molecules is furtherdiscussed in, e.g., Hermanson, Bioconjugate Techniques, Academic PressSan Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al.,“Succinimidyl Carbonates of Polyethylene Glycol,” in Dunn andOttenbrite, eds., Polymeric Drugs and Drug Delivery Systems, AmericanChemical Society, Washington, D.C. (1991).

Conveniently, PEG can be attached to a chosen position in thepolypeptide by site-specific mutagenesis as long as the activity of theconjugate is retained. A target for PEGylation could be any Cysteineresidue at the N-terminus or the C-terminus of the peptide sequence.Additionally or alternatively, other Cysteine residues can be added tothe polypeptide amino acid sequence (e.g., at the N-terminus or theC-terminus) to thereby serve as a target for PEGylation. Computationalanalysis may be effected to select a preferred position for mutagenesiswithout compromising the activity.

Various conjugation chemistries of activated PEG such as PEG-maleimide,PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide canbe employed. Methods of preparing activated PEG molecules are known inthe arts. For example, PEG-VS can be prepared under argon by reacting adichloromethane (DCM) solution of the PEG-OH with NaH and then withdi-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2gram PEG/mL DCM). PEG-AC is made under argon by reacting a DCM solutionof the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM). Suchchemical groups can be attached to linearized, 2-arm, 4-arm, or 8-armPEG molecules.

Resultant conjugated molecules (e.g., PEGylated or PVP-conjugatedpolypeptide) are separated, purified and qualified using e.g.,high-performance liquid chromatography (HPLC) as well as biologicalassays.

According to specific embodiments, the CD44vRA peptide portion of thepolypeptides of the invention other than those listed as consisting ofSEQ ID NOs: 1-3 are 5-100, 5-50, or 5-40, or 5-20, 5-15, 5-10, 5-9, 5-7amino acids in length.

According to specific embodiments, the peptide portion of thepolypeptides of the invention does not comprise a CD44vRA amino acidsequence other than those listed as consisting of SEQ ID NOs: 1-3.

The peptides and compositions of matter of the present invention may beattached (either covalently or non-covalently) to a penetrating agent.

As used herein the phrase “penetrating agent” refers to an agent whichenhances translocation of any of the attached peptide or composition ofmatter across a cell membrane.

According to one embodiment, the penetrating agent is a peptide and isattached to the polypeptide (either directly or non-directly) via apeptide bond.

Typically, peptide penetrating agents have an amino acid compositioncontaining either a high relative abundance of positively charged aminoacids such as lysine or arginine, or have sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids.

According to specific embodiments, the polypeptide is provided in aformulation suitable for cell penetration that enhances intracellulardelivery of the polypeptide as further described hereinbelow.

By way of non-limiting example, cell penetrating peptide (CPP) sequencesmay be used in order to enhance intracellular penetration; however, thedisclosure is not so limited, and any suitable penetrating agent may beused, as known by those of skill in the art.

Cell-Penetrating Peptides (CPPs) are short peptides (≦40 amino acids),with the ability to gain access to the interior of almost any cell. Theyare highly cationic and usually rich in arginine and lysine amino acids.They have the exceptional property of carrying into the cells a widevariety of covalently and noncovalently conjugated cargoes such asproteins, oligonucleotides, and even 200 nm liposomes. Therefore,according to additional exemplary embodiment CPPs can be used totransport the polypeptide or the composition of matter to the interiorof cells.

TAT (transcription activator from HIV-1), pAntp (also named penetratin,Drosophila antennapedia homeodomain transcription factor) and VP22 (fromHerpes Simplex virus) are examples of CPPs that can enter cells in anon-toxic and efficient manner and may be suitable for use with someembodiments of the invention. Protocols for producing CPPs-cargosconjugates and for infecting cells with such conjugates can be found,for example L Theodore et al. [The Journal of Neuroscience, (1995)15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994)91:664-668], and Jing Bian et al. [Circulation Research (2007) 100:1626-1633].

The polypeptides of some embodiments of the invention may be synthesizedby any techniques that are known to those skilled in the art of peptidesynthesis, including solid phase and recombinant techniques.

Any of the proteinaceous polypeptides described herein can be encodedfrom a polynucleotide. These polynucleotides can be used as therapeuticsper se or in the recombinant production of the agent.

Thus, according to an aspect of the present invention there is providedan isolated polynucleotide comprising a nucleic acid sequence encodingthe polypeptide of the present invention.

Thus, according to an aspect of the present invention there is provideda nucleic acid construct comprising the isolated polynucleotide.

Such a nucleic acid construct or system includes at least one cis-actingregulatory element for directing expression of the nucleic acidsequence. Cis-acting regulatory sequences include those that directconstitutive expression of a nucleotide sequence as well as those thatdirect inducible expression of the nucleotide sequence only undercertain conditions. Thus, for example, a promoter sequence for directingtranscription of the polynucleotide sequence in the cell in aconstitutive or inducible manner is included in the nucleic acidconstruct.

The isolated polypeptides and the compositions of matter of the presentinvention are endowed with anti-inflammatory activity.

As used herein, the phrase “anti-inflammatory activity” refers toprevention and/or reduction of acute and/or chronic inflammatoryresponses and/or in preventing and/or treating an inflammatory-relateddisease. Assays for qualifying an anti-inflammatory activity include butare not limited to those described in the Examples section which followsusing both in-vitro and in-vivo models for inflammatory conditions (e.g.RA). Non-limiting examples include paw swelling in vivo in a CIA mousemodel (see e.g. Nedvetzki, et al., (2004) PNAS 101, 18081-18086),histological examination of joint sections obtained from CIA mice andin-vitro cell viability of fibroblasts obtained from synovial fluid ofan RA patient as further described hereinbelow.

According to specific embodiments, the anti-inflammatory activity is notdependent on vaccination or mucosal tolerance.

According to specific embodiments, the isolated polypeptide of thecomposition of matter does not effect immune response in general, as maybe evaluated by a delayed type hypersensitivity assay (DTH) such asdisclosed in Weiss et al., (2000) Proc. Natl. Acad. Sci. USA. 97,285-290; and in Example 5 in the Examples section which follows.

According to specific embodiments, the isolated polypeptide or thecomposition of matter is capable of binding a protein selected from thegroup consisting of serum amyloid A, Transthyretin and apolipoprotein B.

As used herein, the term “serum amyloid A” or “SAA” refer to thepolynucleotide and expression product e.g., polypeptide of the SAA1,SAA2 and SAA4 genes. SAA1 is also known as serum amyloid A1, MGC1 11216,PIG4, SAA, and tumor protein p53 inducible protein 4 (TP53I4). Accordingto specific embodiments the SAA1 refers to the human SAA1, such asprovided in the following GeneBank Numbers NM_199161 and NM_000331 andUniprot Number: P0DJI8 (SEQ ID NOs: 12-14). According to specificembodiments the SAA1 refers to the mouse SAA1, such as provided in thefollowing GeneBank Number NM 009117 (SEQ ID NO: 15). SAA2 is also knownas serum amyloid A2 and SAA. According to specific embodiments the SAA2refers to the human SAA2, such as provided in the following GeneBankNumbers NM_001127380 and NM_030754 and Uniprot number P0DJI8 (SEQ IDNOs: 16-18). According to specific embodiments the SAA2 refers to themouse SAA2, such as provided in the following GeneBank Numbers NM_011314(SEQ ID NO: 19). According to specific embodiments the SAA4 refers tothe human SAA4, such as provided in the following GeneBank NumberNM_006512 and Uniprot Number P35542 (SEQ ID NOs: 20-21).

According to specific embodiments, the term “SAA” refers to SAA1 andSAA2 genes which belong to the serum amyloid A acute phase family ofproteins.

As used herein, the term “Transthyretin”, refers to the polynucleotideand expression product e.g., polypeptide of the TTR gene, which is aprotein carrier of the thyroid hormone thyroxine and retinol. Accordingto specific embodiments the transthyretin refers to the humantransthyretin, such as provided in the following GeneBank NumbersNP_000362 and NM_000371 (SEQ ID NOs: 22-23). According to other specificembodiments, the transthyretin refers to the mouse transthyretin, suchas provided in the following GeneBank Numbers NP_038725 and NM_013697(SEQ ID NOs: 24-25).

As used herein, the term “apolipoprotein B”, refers to thepolynucleotide and expression product e.g., polypeptide of the APOBgene. According to specific embodiments the apolipoprotein B refers tothe human apolipoprotein B, such as provided in the following GeneBankNumbers NP_000375 and NM_000384 (SEQ ID NOs: 26-27). According to otherspecific embodiments, the apolipoprotein B refers to the mouseapolipoprotein B, such as provided in the following GeneBank NumbersNP_033823 and NM_009693 (SEQ ID NOs: 28-29).

By virtue of their anti-inflammatory activity, the polypeptides andcompositions of matter of the present invention may be used to treatdiseases which are dependent on CD44vRA (activity or expression) fortheir onset or progression, such as for the treatment of inflammatorydiseases, such as Rheumatoid Arthritis (RA).

Thus, according to an aspect of the present invention there is provideda method of treating an inflammatory disease in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the isolated polypeptide, thecomposition of matter or the pharmaceutical composition, therebytreating the inflammatory disease in the subject.

According to another aspect of the present invention there is provided ause of the isolated polypeptide, the composition of matter or thepharmaceutical composition for the manufacture of a medicament for thetreatment of an inflammatory disease.

As used herein the term “treating” refers to inhibiting, preventing orarresting the development of a pathology (disease, disorder, orcondition e.g., inflammation e.g., RA) and/or causing the reduction,remission, or regression of a pathology. Those of skill in the art willunderstand that various methodologies and assays can be used to assessthe development of a pathology, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression of apathology.

According to specific embodiments, the term “treating” refers toameliorating symptoms associated with a RA and related diseases,lessening the severity or curing the diseases, or preventing the diseasefrom occurring, preventing the manifestation of symptoms associated withthe disease before they occur, slowing down the progression of thedisease or deteriorating of the symptoms associated therewith, enhancingthe onset of the remission period, slowing down the irreversible damagecaused in the progressive chronic stage of the disease, delaying theonset of said progressive stage, improving survival rate or more rapidrecovery, or a combination of two or more of the above.

As used herein the phrase “subject in need thereof” refers to amammalian male or female subject (e.g., human being) who is diagnosedwith an inflammatory disease or is at risk of to develop an inflammatorydisease. Veterinary uses are also contemplated. The subject may be ofany age including neonatal, infant, juvenile, adolescent, adult andelderly adult.

Methods of determining inflammation in a subject are well known in theart and include, but are not limited to, determining in a blood samplefrom the subject the erythrocyte sedimentation rate (ESR); plasmaviscosity; levels of C-reactive protein (CRP); levels of certaininflammatory cytokines such as IL6 and TNFα; and determination of aninflammation index such as using fibrinogen measurements and hematocritor hemoglobin.

According to specific embodiments, the inflammatory disease involvescells expressing CD44vRA. Non-limiting examples of assays that canevaluate the expression of CD44vRA on cells include flow cytometry andimmunocytochemistry.

Examples of inflammatory diseases (also referred to herein asinflammation or inflammatory condition) include, but not limited to,chronic inflammatory disease and acute inflammatory disease.

Examples for Inflammatory disease include, but not limited toinflammatory diseases associated with hypersensitivity, autoimmunediseases, infectious diseases, graft rejection diseases, allergicdiseases and cancerous diseases.

Inflammatory Diseases Associated with Hypersensitivity

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

Type I or immediate hypersensitivity, such as asthma.

Type II hypersensitivity include, but are not limited to, rheumatoiddiseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V.et al., Histol Histopathol 2000 July; 15 (3):791), Psoriatic Arthritis(PA), spondylitis, ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmunediseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res1998; 17 (1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. etal., Clin Diagn Lab Immunol. 1999 March; 6 (2):156); Chan O T. et al.,Immunol Rev 1999 June; 169:107), glandular diseases, glandularautoimmune diseases, pancreatic autoimmune diseases, diabetes, Type Idiabetes (Zimmet P. Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves' disease(Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29 (2):339),thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and YuS, J Immunol 2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis(Toyoda N. et al., Nippon Rinsho 1999 August; 57 (8):1810), myxedema,idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 August; 57(8):1759); autoimmune reproductive diseases, ovarian diseases, ovarianautoimmunity (Garza K M. et al., J Reprod Immunol 1998 February; 37(2):87), autoimmune anti-sperm infertility (Diekman A B. et al., Am JReprod Immunol. 2000 March; 43 (3):134), repeated fetal loss (Tincani A.et al., Lupus 1998; 7 Suppl 2:S107-9), neurodegenerative diseases,neurological diseases, neurological autoimmune diseases, multiplesclerosis (Cross A H. et al., J Neuroimmunol 2001 Jan. 1; 112 (1-2):1),Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante A J. And Kraig E, Int Rev Immunol1999; 18 (1-2):83), motor neuropathies (Kornberg A J. J Clin Neurosci.2000 May; 7 (3):191), Guillain-Barre syndrome, neuropathies andautoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April; 319(4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome(Takamori M. Am J Med Sci. 2000 April; 319 (4):204), paraneoplasticneurological diseases, cerebellar atrophy, paraneoplastic cerebellaratrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome, polyendocrinopathies, autoimmunepolyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris)2000 January; 156 (1):23); neuropathies, dysimmune neuropathies(Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl1999; 50:419); neuromyotonia, acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135),myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9),granulomatosis, Wegener's granulomatosis, arteritis, Takayasu'sarteritis and Kawasaki syndrome (Praprotnik S. et al., Wien KlinWochenschr 2000 Aug. 25; 112 (15-16):660); anti-factor VIII autoimmunedisease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26(2):157); vasculitises, necrotizing small vessel vasculitises,microscopic polyangiitis, Churg and Strauss syndrome,glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis,crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris) 2000May; 151 (3):178); antiphospholipid syndrome (Flamholz R. et al., J ClinApheresis 1999; 14 (4):171); heart failure, agonist-likebeta-adrenoceptor antibodies in heart failure (Wallukat G. et al., Am JCardiol. 1999 Jun. 17; 83 (12A):75H), thrombocytopenic purpura (MocciaF. Ann Ital Med Int. 1999 April-June; 14 (2):114); hemolytic anemia,autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285), gastrointestinal diseases, autoimmune diseasesof the gastrointestinal tract, intestinal diseases, chronic inflammatoryintestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y.Harefuah 2000 Jan. 16; 138 (2):122), autoimmune diseases of themusculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E.et al., Int Arch Allergy Immunol 2000 September; 123 (1):92); smoothmuscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999June; 53 (5-6):234), hepatic diseases, hepatic autoimmune diseases,autoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326) andprimary biliary cirrhosis (Strassburg C P. et al., Eur J GastroenterolHepatol. 1999 June; 11 (6):595).

Type IV or T cell mediated hypersensitivity, include, but are notlimited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevittH O. Proc Natl Acad Sci U S A 1994 Jan. 18; 91 (2):437), systemicdiseases, systemic autoimmune diseases, systemic lupus erythematosus(Datta S K., Lupus 1998; 7 (9):591), glandular diseases, glandularautoimmune diseases, pancreatic diseases, pancreatic autoimmunediseases, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev.Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves'disease (Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77);ovarian diseases (Garza K M. et al., J Reprod Immunol 1998 February; 37(2):87), prostatitis, autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893), polyglandular syndrome, autoimmunepolyglandular syndrome, Type I autoimmune polyglandular syndrome (HaraT. et al., Blood. 1991 Mar. 1; 77 (5):1127), neurological diseases,autoimmune neurological diseases, multiple sclerosis, neuritis, opticneuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), stiff-man syndrome (Hiemstra H S. et al., ProcNatl Acad Sci USA 2001 Mar. 27; 98 (7):3988), cardiovascular diseases,cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al., J ClinInvest 1996 Oct. 15; 98 (8):1709), autoimmune thrombocytopenic purpura(Semple J W. et al., Blood 1996 May 15; 87 (10):4245), anti-helper Tlymphocyte autoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11(1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 March; 74(3):139), hepatic diseases, hepatic autoimmune diseases, hepatitis,chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol1990 March; 54 (3):382), biliary cirrhosis, primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551), nephricdiseases, nephric autoimmune diseases, nephritis, interstitial nephritis(Kelly C J. J Am Soc Nephrol 1990 August; 1 (2):140), connective tissuediseases, ear diseases, autoimmune connective tissue diseases,autoimmune ear disease (Yoo T J. et al., Cell Immunol 1994 August; 157(1):249), disease of the inner ear (Gloddek B. et al., Ann N Y Acad Sci1997 Dec. 29; 830:266), skin diseases, cutaneous diseases, dermaldiseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoidand pemphigus foliaceus. Note that several same diseases are can beclassified to different classes of hypersensitivity, because theheterogeneity of these diseases.

Examples of delayed type hypersensitivity include, but are not limitedto, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include,but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, butare not limited to, T_(h)1 lymphocyte mediated hypersensitivity andT_(h)2 lymphocyte mediated hypersensitivity.

Autoimmune Diseases

Include, but are not limited to, cardiovascular diseases, rheumatoiddiseases, glandular diseases, gastrointestinal diseases, cutaneousdiseases, hepatic diseases, neurological diseases, muscular diseases,nephric diseases, diseases related to reproduction, connective tissuediseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660), anti-factorVIII autoimmune disease (Lacroix-Desmazes S. et al., Semin ThrombHemost. 2000; 26 (2):157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne(Paris) 2000 May; 151 (3):178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999; 14 (4):171), antibody-induced heartfailure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17; 83 (12A):75H),thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 April-June;14 (2):114; Semple J W. et al., Blood 1996 May 15; 87 (10):4245),autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March; 74(3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al.,J Clin Invest 1996 Oct. 15; 98 (8):1709) and anti-helper T lymphocyteautoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11 (1):9).

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July;15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. Diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res ClinPract 1996 October; 34 Suppl: S125), autoimmune thyroid diseases,Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June;29 (2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March; 92(1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S,J Immunol 2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (ToyodaN. et al., Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema(Mitsuma T. Nippon Rinsho. 1999 August; 57 (8):1759), ovarianautoimmunity (Garza K M. et al., J Reprod Immunol 1998 February; 37(2):87), autoimmune anti-sperm infertility (Diekman A B. et al., Am JReprod Immunol. 2000 March; 43 (3):134), autoimmune prostatitis(Alexander R B. et al., Urology 1997 December; 50 (6):893) and Type Iautoimmune polyglandular syndrome (Hara T. et al., Blood. 1991 Mar. 1;77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) andautoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E,Int Rev Immunol (1999) 18(1-2):83; Oshima M. et al., Eur J Immunol(1990) 20(12):2563), neuropathies, motor neuropathies (Kornberg A J. JClin Neurosci. (2000) 7(3):191); Guillain-Barre syndrome and autoimmuneneuropathies (Kusunoki S. Am J Med Sci. (2000) 319(4):234), myasthenia,Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. (2000)319(4):204); paraneoplastic neurological diseases, cerebellar atrophy,paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra H S.et al., Proc Natl Acad Sci units S A (2001) 98(7):3988);non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies,encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis,Sydeham chorea, Gilles de la Tourette syndrome and autoimmunepolyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris)2000 January; 156 (1):23); dysimmune neuropathies (Nobile-Orazio E. etal., Electroencephalogr Clin Neurophysiol Suppl 1999; 50:419); acquiredneuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al.,Ann N Y Acad Sci. 1998 May 13; 841:482), neuritis, optic neuritis(Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May; 57(5):544) and neurodegenerative diseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother1999 June; 53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol 1990 August; 1 (2):140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7Suppl 2:S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol 1994 August; 157 (1):249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107).

Infectious Diseases

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

Graft Rejection Diseases

Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyper-acute graft rejection, acute graftrejection and graft versus host disease.

Allergic Diseases

Examples of allergic diseases include, but are not limited to, asthma,hives, urticaria, pollen allergy, dust mite allergy, venom allergy,cosmetics allergy, latex allergy, chemical allergy, drug allergy, insectbite allergy, animal dander allergy, stinging plant allergy, poison ivyallergy and food allergy.

Cancerous Diseases

Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma, and leukemia. Particular examples of cancerousdiseases but are not limited to: Myeloid leukemia such as Chronicmyelogenous leukemia. Acute myelogenous leukemia with maturation, Acutepromyelocytic leukemia, Acute nonlymphocytic leukemia with increasedbasophils, Acute monocytic leukemia. Acute myelomonocytic leukemia witheosinophilia; Malignant lymphoma, such as Burkitt's Non-Hodgkin's;Lymphocytic leukemia, such as Acute lymphoblastic leukemia. Chroniclymphocytic leukemia; Myeloproliferative diseases, such as Solid tumorsBenign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus,Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovialsarcoma, Rhabdomyosarcoma (alveolar), Extraskeletal myxoidchonodrosarcoma, Ewing's tumor; other include Testicular and ovariandysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignantmelanoma, Mesothelioma, breast, skin, prostate, and ovarian.

According to specific embodiments the inflammatory disease is selectedfrom the group consisting of Rheumatoid arthritis (RA), psoriaticarthritis, Alzheimer's disease, cancer and cardiovascular disease.

According to other specific embodiments the inflammatory diseasecomprises RA.

As used herein, the phrase “rheumatoid arthritis (RA)” refers to anautoimmune disease which primarily affects the joints. RA includes, butis limited to, adult RA, juvenile idiopathic arthritis, juvenile RA andjuvenile chronic arthritis. RA can be diagnosed according to theAmerican Rheumatoid Association criteria for the classification ofrheumatoid arthritis, or any similar criteria and includes active, early(active RA diagnosed for at least 8 weeks but no longer than four years)and incipient (polyarthritis that does not fully meet the criteria for adiagnosis of RA, in association with the presence of RA-specificprognostic biomarkers such as anti-CCP and shared epitope) RA.

Example of RA clinical parameters symptoms that can be monitored toindicate improvement during treatment with the isolated polypeptides andcomposition of matter of the invention are:

Morning stiffness for at least one hour and present for at least sixweeks;

Swelling of three or more joints for at least six weeks;

Swelling of wrist, metacarpophalangeal, or proximal interphalangealjoints for at least six weeks

Symmetric joint swelling;

Hand x-ray changes that include erosions or unequivocal bonydecalcification;

Rheumatoid subcutaneous nodules; and

Rheumatoid factors

Additional parameters that can be used in human for assessing RAimprovement can be according to the American College of Rheumatology(ACR) and include e.g. ACR improvement criteria—ACR20, ACR50 and ACR70representing the percentage of disease activity improvement (20, 50 or70%) by the reduction in certain RA symptoms. Thus, for example, ACR20refers to patients which achieve a 20% improvement in tender and swollenjoint counts and 20% improvement in three of the five remaining ACR coreset measures.

As the present inventors discovered 3 potential target proteins of theisolated polypeptides and have also shown that addition of one of theseproteins (i.e. SAA) can prevent the in-vitro activity of the polypeptide(see Examples 7 and 9 and FIG. 15 in the Examples section which follows)the present invention contemplates the use of a combined treatmentcomprising the isolated polypeptides or the compositions of matter ofthe present invention and serum amyloid A (SAA), thranthyretin and/orapolipoproteins B inhibitors.

Thus, according to another aspect of the present invention there isprovided a method of treating an inflammatory disease in a subject inneed thereof, the method comprising:

(a) administering to the subject a therapeutically effective amount ofthe isolated polypeptide or the composition of matter of someembodiments of the present invention; and

(b) administering to said subject a therapeutically effective amount ofan inhibitor of a protein selected from the group consisting of serumamyloid A (SAA), thranthyretin and apolipoproteins B,

thereby treating the inflammatory disease in the subject.

According to another aspect there is provided a use of the isolatedpolypeptide or the composition of matter of some embodiments of thepresent invention and an inhibitor of a protein selected from the groupconsisting of serum amyloid A (SAA), thranthyretin and apolipoprotein Bfor the manufacture of a medicament for the treatment of an inflammatorydisease.

According to another aspect of the present invention there is providedan article of manufacture or a kit identified for use in treatinginflammatory disease, comprising a packaging material packaging theisolated polypeptides or the composition of matter of some embodimentsof the present invention and an inhibitor for a protein selected fromthe group consisting of SAA, thranthyretin and apolipoproteins B.

According to another aspect of the present invention there is provided amethod of treating an inflammatory disease in a subject in need thereof,the method comprising:

(a) administering to the subject a therapeutically effective amount ofan isolated polypeptide comprising an amino acid sequence selected formthe group consisting of SEQ ID NOs: 1-3, wherein said polypeptidecomprises an anti-inflammatory activity; and

(b) administering to said subject a therapeutically effective amount ofan inhibitor of a protein selected from the group consisting of serumamyloid A (SAA), thranthyretin and apolipoproteins B,

thereby treating the inflammatory disease in the subject.

According to another aspect there is provided a use of as isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-3, wherein said polypeptide comprises ananti-inflammatory activity; and an inhibitor of a protein selected fromthe group consisting of serum amyloid A (SAA), thranthyretin andapolipoprotein B for the manufacture of a medicament for the treatmentof an inflammatory disease.

According another aspect of the present invention there is provided anarticle of manufacture or a kit identified for use in treatinginflammatory disease, comprising a packaging material packaging theisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-3, wherein said polypeptide comprisesan anti-inflammatory activity; and an inhibitor for a protein selectedfrom the group consisting of SAA, thranthyretin and apolipoproteins B.

As used herein the term “inhibitor” refers to an agent whichdownregulates expression and/or activity of a protein (e.g. SAA,thranthyretin and apolipoprotein B) at the genomic (e.g. homologousrecombination and site specific endonucleases) and/or the transcriptlevel using a variety of molecules which interfere with transcriptionand/or translation (e.g., RNA silencing agents e.g. siRNA, shRNA,micro-RNA) or on the protein level (e.g., aptamers, small molecules andinhibitory peptides, antagonists, enzymes that cleave the polypeptide,antibodies and the like).

Non-limiting example include an antibody which inhibits SAA such asAnti-serum albumin A, an RNA interference targeted to SAA mRNA (see e.g.International Publication Application No: WO 2006071691), an antisenseoligonucleotides targeted to apolipoprotein B, such as but not limitedto Mipomersen (ISIS-301012, KYNAMRO™), Triazolones as apolipoprotein Bsynthesis inhibitors (see e.g. U.S. Pat. No. 6,197,972) and anapolipoprotein B secretion inhibitor (see e.g. EP ApplicationPublication No. EP 1099438).

According to a specific embodiment, step (a) is effected prior to step(b).

According to another specific embodiment, step (a) is effected followingstep (b).

According to yet another specific embodiment, step (a) is effectedconcomitantly with step (b).

Multiple rounds of administration according to the methods of thepresent invention and multiple doses of the isolated polypeptide or thecomposition of matter and the inhibitor can be administered. Accordingto specific embodiments step (a) is effected multiple times. Thus,according to specific embodiments, administration of inhibitor iseffected following at least one administration of the isolatedpolypeptide or the composition of matter. According to specificembodiments step (B) is effected multiple times. Thus, according tospecific embodiments, administering the isolated polypeptide or thecomposition of matter of the present invention is effected following atleast one administration of the inhibitor. According to specificembodiments, administering the isolated polypeptide or the compositionof matter of the present invention is effected in a sequential orderwith administration of the inhibitor.

The isolated polypeptide or the composition of matter and the inhibitormay be packaged in the same container or in separate containers; eachpossibility represents a separate embodiment of the present invention.

According to specific embodiments, the isolated polypeptide or thecomposition of matter and the inhibitor are in separate formulations.

According to other specific embodiments, the isolated polypeptide or thecomposition of matter and the inhibitor are in a co-formulation.

The isolated polypeptides, the compositions of matter and the inhibitorsof the present invention can be provided to the subject per se, or aspart of a pharmaceutical composition where it is mixed with apharmaceutically acceptable carrier.

Thus, according to an aspect of the present invention there is provideda pharmaceutical composition comprising as an active agent the isolatedpolypeptide or the composition of matter; and a pharmaceuticallyacceptable carrier or diluent.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the polypeptide orcomposition of matter comprising the polypeptide accountable for thebiological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,topical, intradermal, rectal, transmucosal, especially transnasal,intestinal or parenteral delivery, including intramuscular, subcutaneousand intramedullary injections as well as intrathecal, directintraventricular, intracardiac, e.g., into the right or left ventricularcavity, into the common coronary artery, intravenous, intraperitoneal,intranasal, or intraocular injections.

According to specific embodiments the route of administration is oraladministration.

According to other specific embodiments, the route of administration isinto the skin. Methods of administering an active agent into a skin areknown in the art and include, for example, intradermal injections, gels,liquid sprays and patches which comprise the active agent and which areapplied on the outer surface of the skin.

According to some embodiments of the invention, administration of theactive agent into the skin of the subject is performed topically (on theskin).

According to some embodiments of the invention, administration of theactive agent into the skin of the subject is performed non-invasively,e.g., using a gel, a liquid spray or a patch (e.g. reservoir type patchand matrix type patch) comprising the active ingredient, which areapplied onto the skin of the subject.

It should be noted that in order to increase delivery of the activeagent into the skin, the active agent can be formulated with variousvehicles designed to increase delivery to the epidermis or the dermislayers. Such vehicles include, but are not limited to liposomes,dendrimers, noisome, transfersome, microemulsion and solid lipidnanoparticles.

According to some embodiments of the invention, administering the isperformed by an intradermal injection.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patientsuch as a local injection into the joint. Methods of administering anactive agent into the joint are known in the art and includeintra-articular injection wherein a hypodermic needle is inserted intothe joint to thereby deliver the active agent to the intra-articularspace of the intra-articular joint.

As described the polypeptides and compositions of matter of theinvention may be used to treat e.g. Alzheimer's disease. Conventionalapproaches for drug delivery to the central nervous system (CNS)include: neurosurgical strategies (e.g., intrahippocampal (IH),intracranial (IC), intracerebral injection, intracerebroventricularinjection (ICV) or infusion or intrathecal administration); molecularmanipulation of the agent (e.g., production of a chimeric fusion proteinthat comprises a transport peptide that has an affinity for anendothelial cell surface molecule in combination with an agent that isitself incapable of crossing the BBB) in an attempt to exploit one ofthe endogenous transport pathways of the BBB; pharmacological strategiesdesigned to increase the lipid solubility of an agent (e.g., conjugationof water-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Pharmaceutical compositions of some embodiments of the invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodimentsof the invention thus may be formulated in conventional manner using oneor more physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

According to specific embodiments, the pharmaceutical composition isformulated for oral administration.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to some embodiments of the invention are convenientlydelivered in the form of an aerosol spray presentation from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of some embodiments of the invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

The pharmaceutical composition of some embodiments of the invention mayalso be formulated for sustained-release to provide elevated serumhalf-life. Such sustained release systems are well known to those ofskill in the art and include e.g. microcapsules and nanoparticles.According to specific embodiments, the ProLease biodegradablemicrosphere delivery system for proteins and peptides (Tracy, 1998,Biotechnol. Prog. 14, 108; Johnson et al., 1996, Nature Med. 2, 795;Herbert et al., 1998, Pharmaceut. Res. 15, 357) a dry powder composed ofbiodegradable polymeric microspheres containing the protein in a polymermatrix that can be compounded as a dry formulation with or without otheragents.

Pharmaceutical compositions suitable for use in context of someembodiments of the invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, a therapeutically effective amount means anamount of active ingredients effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., RA) or prolong the survival ofthe subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide thatthe levels of the active ingredient are sufficient to induce or suppressthe biological effect (minimal effective concentration, MEC). The MECwill vary for each preparation, but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

The doses shown herein with respect to the mouse animal model can beconverted for the treatment other species such as human and otheranimals diagnosed with the inflammatory disease. Conversion Tableapproved by the FDA is shown in Reagan-Shaw S., et al., FASEB J.22:659-661 (2007).

The human equivalent dose is calculated as follows: HED (mg/kg)=Animaldose (mg/kg) multiplied by (Animal K_(m)/human K_(m)).

According to some embodiments of the invention, the isolated polypeptideor the composition of matter is provided at an amount equivalent to arange of from about 2.5-40 mg/kg/day in mice, including any intermediatesubranges and values therebetween.

According to specific embodiments the isolated polypeptide or thecomposition of matter is provided at an amount equivalent to about 3.5mg/kg/day in mice.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

It will be appreciated that the therapeutic agents of the presentinvention can be provided to the individual with additional activeagents to achieve an improved therapeutic effect as compared totreatment with each agent by itself. In such therapy, measures (e.g.,dosing and selection of the complementary agent) are taken to adverseside effects which may be associated with combination therapies.

Administration of such combination therapy can be simultaneous, such asin a single capsule having a fixed ratio of these active agents, or inmultiple capsules for each agent.

Thus, the agents of the present invention can be administered alone withother established or experimental therapeutic regimen to treatinflammatory disease such as nonsteroidal anti-inflammatory drugs(NSAID), disease-modifying antirheumatic drugs (DMARDS),corticosteroids, analgesics, Fibromyalgia medications, chemotherapeuticagents and other treatment regimens which are well known in the art.

As mentioned, the present inventors have developed a novel in-vitroassay to test the activity of the peptides and compositions of matter ofthe present invention. The assay is based on the realization that the RApeptides can reduce survival of fibroblasts isolated from the synovialfluid of an RA patient. Thus, this assay can be used to compare batch tobatch variation of manufactured peptides and compositions of matterpeptides of the present invention for qualifying the anti-inflammatoryactivity as well as for testing e.g. stability of the peptides andcompositions of matter of the present invention following exposure toenvironmental conditions such as storage temperature, modifications tothe peptides and the formulations.

Thus, according to an aspect of the present invention there is provideda method of determining potency of a batch of the isolated polypeptide,the composition of matter or the pharmaceutical composition of someembodiments of the present invention, the method comprising:

(a) contacting a batch of the isolated polypeptide, the composition ofmatter or the pharmaceutical composition with fibroblasts obtained froman inflammatory joint of a Rheumatoid arthritis patient; and

(b) determining survival of said fibroblasts following a predeterminedincubation time, so as to determine the potency of the batch.

According to specific embodiments, the method comprising synthesizingthe isolated polypeptide, the composition of matter or thepharmaceutical composition with a modification. Such a modification canbe any of the modifications presented hereinabove.

According to specific embodiments, the method is effected in-vitro orex-vivo.

As used herein, the term “potency” refers to the measure of thebiological activity of the product (i.e.; the isolated polypeptide orthe composition of matter), based on the attribute of the product whichis linked to the relevant biological properties (i.e.; reduced survivalof fibroblasts obtained from an RA patient).

As used herein, the term “batch” refers to a specific quantity of a drugthat is intended to have uniform character and quality, within specifiedlimits, and is typically produced according to a single manufacturingorder during the same cycle of manufacture. Thus, the present teachingscan be used in the QA of the manufacturing procedures for assessing thebiological activity of the isolated polypeptides, the composition ofmatter or the pharmaceutical compositions as part of batchqualification.

According to specific embodiments, the term “batch” also refers to aquantity of the drug exposed to stability characterization and/orpeptide and formulation modifications.

As used herein, the term “fibroblast” refers to a connective tissue cellthat synthesizes the extracellular matrix and collagen and is obtainedfrom a synovial fluid of an RA patient. The fibroblasts used accordingto the method can be a primary culture directly isolated from an RApatient or cell lines obtained from the fibroblasts such as thecommercially available cell lines SW 982, PCS-201-010 and ACS-1023 thatcan be obtained from the ATCC.

Methods for obtaining a synovial fluid from a subject are well known inthe art and include, but are not limited to biopsy such as joint biopsyand joint aspiration. Typically, procedures for obtaining tissue orfluid biopsies are described in details in Hypertext TransferProtocol://World Wide Web (dot) healthatoz(dot)com/healthatoz/Atoz/search.asp.

Specifically, a joint aspiration, also known as Arthrocentesis refers tothe removal of fluid from the space around a joint using a needle andsyringe. This is usually performed under a local anesthetic to eitherrelieve swelling or to obtain fluid for analysis to diagnose a jointdisorder and/or problem. Joint aspiration is usually performed on theknee; however, fluid can also be removed from other joints, such as thehip, ankle, shoulder, elbow, or wrist.

A joint biopsy refers to joint or synovial biopsy. In the procedure asample of the joint lining or synovial membrane or fluid is taken.Briefly, the procedure is effected in a clinical facility by a surgeon.A number of approaches are available to perform this biopsy: such asthrough an incision in the joint; with a scope inserted in the joint;or, more typically, by the insertion of a sharp instrument through theskin. The sample can be taken from any joint, typically the examinedjoint is the knee. A sharp instrument (trocar) is pushed into the jointspace. A needle with an attached syringe is inserted into the joint towithdraw fluid for laboratory analysis. The surgeon may instillanalgesic compounds into the joint and along the needle track before theneedle is withdrawn. The trocar and then the biopsy needle is insertedand specimens taken. After the specimen is taken, both the trocar andthe biopsy needle are removed.

Regardless of the procedure employed, once the biological sample isobtained, the fibroblast may be further isolated. Enrichment offibroblasts populations can be obtained by methods well known in theart, and included those disclosed in e.g. Bendersky et al, J Immunol.;188:4349-59, 2012, magnetic cell separation and flow cytometry cellsorting.

Thus, for example the fibroblasts can be isolated by culturing adherentsynovial fluid cells in plastic wells in DMEM-supplemented medium forabout 48 hours followed by removal of all of the nonadherent cells. Theadherent cells which comprise fibroblasts typically display afibroblastic morphology and express CD90, low levels of the CD49aintegrin, negative for macrophage (CD14), T cell (CD3), and B cell(CD20) surface markers (as determined by e.g. flow cytometry) and stainpositively for collagen type I determined by Sirius red staining.

The primary culture of fibroblasts comprises according to specificembodiments at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% ormore fibroblasts.

Following preparation of the fibroblasts, predetermined amount of cellsare incubated in tissue culture plates (e.g.; 12, 24, 96, 384 wellsplates) with the appropriate Growth medium and stimulated with apredetermined amount of the tested isolated polypeptide, the compositionof matter or the pharmaceutical composition. Selection of the medium iswell within the capabilities of skilled in the art. Thus, for example,RPMI or DMEM (can be obtained for example from Sigma-Aldrich orBiological Industries, Beit Haemek, Israel) can be used as a growthmedium. The medium may be supplemented with L-glutamine, non-essentialamino acids, sodium pyruvate, antibiotic/antimycotic solution,2-mercaptoethanol and serum.

Selection of the predetermined amount of cells incubated for in-vitrotesting that will result in detectable effect on cell survival is wellwithin the capabilities of the skilled in the art. Thus, for examplecell concentration can be 1×10⁴/ml to 5×10⁶/ml; 1×10⁴/ml to 1×10⁶/ml;5×10⁴/ml to 5×10⁶/ml; 1×10⁵/ml to 5×10⁶/ml; or 1×10⁵/ml to 1×10⁶/ml.

According to specific embodiments the cell concentration is 2×10⁵/ml.

Selection of the peptide or composition of matter concentration used forthe in-vitro testing that will result in detectable effect on cellsurvival is well within the capabilities of skilled in the art.Preferably, the concentration used should be within the linear range ofthe selected stimulation parameter. Thus, for example, the concentrationcan be 1 μg/ml to 30 μg/ml; 1 μg/ml to 20 μg/ml; 5 μg/ml to 30 μg/ml; or5 μg/ml to 20 μg/ml.

The number of tested concentration can be at least 1, at least 2, atleast 3, at least 5, at least 6, 1-10, 2-10, 3-10, 5-10, 1-5, 2-5 and3-5 different concentrations.

The number of samples repeats for each of the tested concentration canbe 2, 3, 4, 5 or 6 repeats.

Following a pre-determined incubation time the survival of thefibroblasts is determined. Specific methods of monitoring cell survivalare known in the art and include for example, the MTT test which isbased on the selective ability of living cells to reduce the yellow saltMTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (canbe obtained for example from Sigma, Aldrich) to a purple-blue insolubleformazan precipitate; Apoptosis assays such as the TUNEL assay (can beobtained for example from Roche); and the Annexin V assay [for exampleApoAlert® Annexin V Apoptosis Kit (Clontech Laboratories, Inc., CA,USA)].

The incubation time may vary and determination of the incubation timethat will result in detectable effect on cell survival is well withinthe capabilities of skilled in the art. According to a specificembodiment, the incubation time is between 12 hours to 96 hours.According to some embodiments of the invention, the incubation time isbetween 12 to 72 hours; 12 to 48 hours; 12 to 24 hours; 24 to 96 hours,24 to 72 hours or 24 to 48 hours. According to specific embodiments ofthe invention, the incubation time is between 24-48 hours.

According to specific embodiments, reduced survival of the fibroblastsfollowing said contacting is indicative that the batch is potent.

According to specific embodiments the assay may further include positiveand negative control samples. The positive control for the assay mayinclude agents inducing non-specific fibroblasts cell death, for exampleascorbate (see e.g. Schmidt et al., J Biomed Mater Res. 1993 April;27(4):521-30).

Negative control for the assay may include agents which prevent thebiological activity of the tested polypeptide or composition of mattersuch as SAA (can be obtained for example from PeproTech).

According to specific embodiments, the method comprising comparing thesurvival of the cells with survival of the cells following contactingwith a reference standard batch of the isolated polypeptide, compositionof matter or the pharmaceutical composition, so as to determine therelative potency of the batch.

As used herein, the term “relative potency” refers to a qualitativemeasure of potency of a batch of the isolated polypeptide, compositionof matter or the pharmaceutical composition, relatively to a standardreference (RS) of the isolated polypeptide, composition of matter or thepharmaceutical composition, having a known potency.

According to specific embodiments the potency of a batch of the isolatedpolypeptide, composition of matter or the pharmaceutical composition, isdetermined relatively to the known potency of a reference standard (RS).

As used herein, the phrase “reference standard” or “RS” refers to astandardized isolated polypeptide, composition of matter orpharmaceutical composition, which is used as a measurement base for theisolated polypeptide, composition of matter or the pharmaceuticalcomposition. RS provides a calibrated level of biological effect againstwhich new preparations of the isolated polypeptide, composition ofmatter or the pharmaceutical composition can be compared to.

According to a specific embodiment, the RS is characterized by optimumpotency and quality of an active component that is effective in treatingthe disease (e.g., RA).

Calculating potency and relative potency are known in the art. Accordingto specific embodiments the relative potency is calculated using asoftware suitable for biological assays, such as parallel line analysissoftware e.g., PLA (Stegmann Systems GmbH) and Gen5 data analysissoftware (BioTek).

Implementation of the method and/or system of embodiments of theinvention can involve performing or completing selected tasks manually,automatically, or a combination thereof. Moreover, according to actualinstrumentation and equipment of embodiments of the method and/or systemof the invention, several selected tasks could be implemented byhardware, by software or by firmware or by a combination thereof usingan operating system.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Peptide Synthesis and Characterization

Materials and Methods

Synthesis of 5, 7 and 9 Mer Peptides—

The 5-, 7- and 9-mer peptides [MTADV (SEQ ID NO: 1), MTADVDR (SEQ ID NO:2) and TRMTADVDR (SEQ ID NO: 3)], −3, the Acylated-N and Amidated-Ctermini 5-, 7- and 9-mer peptides [Ac-MTADV-NH2 (SEQ ID NO: 4),Ac-MTADVDR-NH₂ (SEQ ID NO: 5) and Ac-TRMTADVDR-NH₂ (SEQ ID NO: 6)] andthe scrambled 7-mer peptide [Ac-TMDVADR-NH₂ (SEQ ID NO: 7)], weresynthesized by Sigma Israel using solid phase synthesis fmoc chemistry.A purity of 95- to 97% was reached.

Liquid Chromatography-Mass Spectrometry (LCMS)—

Stability, Pharmacokinetic (PK) and target proteins of the peptide wereevaluated by LCMS.

Sample Preparation for Mass Spectrometry:

The protein-bound beads were reduced with 2.8 mM DTT (60° C. for 30 min)and modified with 8.8 mM iodoacetamide (at room temperature for 30 minunder dark conditions) in 8M Urea and 400 mM Ammonium bicarbonate. Theproteins were digested in 2M Urea, 25 mM ammonium bicarbonate withmodified trypsin (Promega) at a 1:50 enzyme-to-substrate ratio,overnight at 37° C. The tryptic peptides were desalted using C18 tips(Harvard) dried and re-suspended in 0.1% Formic acid Mass Spectrometrymeasurements. The resulting tryptic peptides were analyzed by LC-MS/MSusing an OrbitrapXL mass spectrometer (Thermo-Fisher) fitted with acapillary HPLC (Eksigent). Specifically, the peptides were loaded onto aC18 trap column (0.3 5 mm, LC-Packings) connected on-line to a homemadecapillary column (75 micron ID) packed with Reprosil C18-Aqua (Dr MaischGmbH, Germany) and resolved using linear 94 minutes 5 to 40%acetonitrile gradients followed by 12 minutes at 95% acetonitrile in thepresence of 0.1% formic acid in water at flow rates of 0.25 μl/min. Massspectrometry was performed in a positive mode using repetitively full MSscan (resolution 60000) followed by collision induces dissociation(CID). Top seven, (>1 charged peptides, 350-2000 M/Z) were selected forfragmentation from each full mass spectrum.

Data Analysis:

The mass spectrometry was analyzed using the Discoverer software version1.4 against the human uniprot database and against decoy databases (inorder to determine the false discovery rate (FDR), using the Sequest andMascot search engines. High confidence refers to 0.01 FDR.

Semi quantitation was performed by calculating the peak area of eachpeptide. The area of the protein is the average of the three mostintense peptides from each protein.

Results

Inclusion of alanine in the splicing junction between variant exon 4 andvariant exon 5 of CD44vRA variant leading to the presence of the MTADVsequence instead of the original MTDV sequence was shown to confer thepathological activity of CD44vRA (Nedvetzki et al., J Clin Invest111:1211-1220, 2003).

The present inventors have synthesized 5-, 7-, and 9-mer peptides(denoted herein as RA peptides) including the MTADV sequence. The 5-merRA peptide comprises a relative hydrophobic amino acid sequence as wellas a low molecular weight (less than 700 Dalton). Therefore, it mayeasily penetrate into tissues and consequently may be used in topicalapplications and oral delivery.

The proteolytic analysis of the RA peptides demonstrated no proteolyticsites (data not shown), indicating that the peptides are at leastrelatively stable. Quantification analysis by Mass Spectrometryindicated almost identical stability of the 5-mer peptide (SEQ ID NO: 1)upon storage in saline at 4° C., at room temperature or at −20° C. for22 weeks (FIG. 1). Assuming that storage at −20° C. displays 100%stability, these results demonstrate that the 5-mer peptide is stablefor at least 22 weeks at room temperature and 4° C.

Example 2 The 5- and 9-Mer RA Peptides can Reduce Joint Inflammation inthe Collagen-Induced Arthritis (CIA) Mouse Model

Materials and Methods

Mice—

Collagen-induced arthritis (CIA) was generated in DBA/1 or C57BL mice byinjection of type II collagen as described in Nedvetzki et al., PNAS101, 18081-18086, 2004.

Treatment Protocol—

PBS, or the 9-mer RA peptide (SEQ ID NO: 3) at a dose of 25 μg, 100 μgor 150 μg per injection was administered i.p to CIA mice for 4 timeswithin 6 days (see Table 3 below). PBS (n=7), or 5-mer RA peptide (SEQID NO: 1, n=7) at a dose of 70 μg per injection were administered i.p toCIA mice on C57BL/6 background for 10 consecutive days. In all groupsfirst injection was given at the onset of disease as determined by pawswelling.

Evaluation of Joint Inflammation—

Inflammation was evaluated by the paw swelling response. Paw swellingwas measured by micro caliber. Paw swelling at range of 2.1-2.3 mm wasconsidered “onset of disease” and the starting point for injection ofthe peptides. Mice that showed above 2.3 mm paw swelling were excludedfrom the experiment. All measurements were performed under blind manner(For additional details see Nedvetzki, et al., (2004) PNAS 101,18081-18086).

Histology—

Mice were sacrificed on day 11 (one day following cessation oftreatment) and hind limbs were isolated and fixed overnight with 4%paraformaldehyde at room temperature (for additional details seeNedvetzki et al. (2004) PNAS 101, 18081-18086). Following fixation,diarthrodial Joint sections were prepared, stained with Hematoxylin andEosin (H&E) and evaluated by a pathologist under blind manner. Jointinfiltration score: 0=no infiltration; 4=massive infiltration.

Statistical Analysis—

Statistical analysis for each measurement point was performed by theStudent's t-test for unpaired values.

TABLE 3 Experimental design-dose response (FIGS. 2A-C) Group # Treatment#Mice Injection protocol 1 1XPBS 100 μl/injection 8 Injection everyother day for 6 constitutive days 2 Peptide 9 mer TRMTADVDR 8 Injectionevery other day (SEQ ID NO: 3) for 6 constitutive days 25 μg/100μl/injection 3 Peptide 9 mer TRMTADVDR 6 Injection every other day (SEQID NO: 3) for constitutive days 100 μg/100 μl/injection 3 Peptide 9 merTRMTADVDR 4 Injection every other day (SEQ ID NO: 3) for 6 constitutivedays 150 μg/100 μl/injection

Results

The collagen-induced arthritis (CIA) mouse model is the mouse analogueof human Rheumatoid Arthritis (RA). To evaluate the effect of thegenerated RA peptides on joint inflammation in the CIA mouse modeldifferent dosages of the 9-mer RA peptide (SEQ ID NO: 3) wereadministered to CIA mice on DBA/1 background starting from the onset ofdisease; and joint inflammation was evaluated by determining pawswelling. As shown in FIGS. 2A-B, mice that received 25 and 100 μg ofthe 9-mer RA peptide (SEQ ID NO: 3) did not show significant differencein paw swelling response as compared to mice administered with PBS,implying lack of a significant therapeutic effect. In contrast, micethat received 150 μg of the 9-mer RA peptide (SEQ ID NO: 3) haddemonstrated significant reduction in paw swelling (FIG. 2C).

Histological evaluation of hind limbs sections taken from CIA mice onC57BL/6 background following treatment with the PBS control revealedmassive infiltration of mononuclear inflammatory cells into the jointcapsule, extensive synovial hypertrophy and severe narrowing of thejoint space which was filled with reactive cells. In addition, severeerosion of the articular cartilage and the subchondral bone, withdestruction of cartilage matrix, was noted. The remaining cartilagematrix was heavily infiltrated as well. Taken together, the averagejoint Infiltration score in the PBS treated mice=about 4 (FIGS. 3A and3C).

On the contrary, histological evaluation of hind limbs sections takenfrom CIA mice following treatment with the 5-mer RA peptide (SEQ IDNO: 1) revealed none-to-mild infiltration of mononuclear inflammatorycells into the joint capsule with either no or mild synovial hypertrophyInflammatory infiltrate was noted in minority of the samples. The jointspace was preserved, with few reactive cells if any. In addition, noerosion of the articular cartilage and the subchondral bone was notedand the cartilage matrix was generally preserved. Furthermore, few ofthe samples were indistinguishable from unaffected mice (data notshown). Taken together, average joint infiltration score in the 5-mer RApeptide treated mice=about 1 (FIGS. 3B and 3C).

Overall, the joint inflammation score of the control PBS treated groupwas significantly higher (P<0.0001) than that of the 5-mer RA peptide(SEQ ID NO: 1) treated group, indicating that treatment with the peptidecan highly restore normal histology of the inflamed joint.

Taken together, treatment with the 5- and 9-mer RA peptides (SEQ ID NOs:1 and 3) inhibit joint inflammation in the CIA mouse model.

Without being bound by theory it is suggested that an interactionbetween unknown CD44vRA ligand and CD44vRA create a conformationalchange of CD44vRA glycoprotein. This new epitope allows binding of FGF-2to the heparin sulfate of v3 exon product resides in the same molecule.The bound FGF-2 is oriented to interact with endothelial cells orfibroblasts expressing FGF receptor 1, resulting in their proliferationand exaggeration of the inflammatory activity (Nedvetzki et al., J ClinInvest 111:1211-1220, 2003). The RA peptide may compete with the cellsurface CD44 on the interaction with the unknown ligand resulting inblockade of the FGF-2-induced inflammatory cascade described byNedvetzki et al. (J Clin Invest 111:1211-1220, 2003).

Example 3 The 5-7- and 9-Mer Protected RA Peptides can Reduce JointInflammation in the CIA Mouse Model

Materials and Methods

Mice—

As described in Example 2 above.

Treatment Protocol—

PBS, or the tested peptides [Ac-TRMTADVDR-NH2 (SEQ ID NO: 6),Ac-MTADVDR-NH2 (SEQ ID NO: 5), Ac-MTADV-NH₂ (SEQ ID NO: 4) and scrambledAc-TMDVADR-NH₂ (SEQ ID NO: 7)] at doses of 70 or 200 μg per injectionwere administered i.p to CIA/1 mice on DBA background for 9-10consecutive days (see Tables 4-7 below). As a non-specific control,several mice were administered I.P. with Dexamethsone (Dex) at a dose of50 μg, following the same protocol. In all groups first injection wasgiven at the onset of disease as determined by paw swelling.

Evaluation of Joint Inflammation—

As described in Example 2 above. Paw swelling of less than 2 mm wasconsidered healthy.

Statistical Analysis—

As described in Example 2 above.

TABLE 4 Experimental design (FIG. 4) Group #Mice/ Injection # Treatmentpaws* protocol 1 PBSx1 100 μl/injection 4/5* One injection per day for 9constitutive days 2 Peptide 9 mer Ac-TRMTADVDR- 5/7* One injection NH2(SEQ ID NO: 6) per day for 9 200 μg/100 μl/injection constitutive days 3Peptide 7 mer Ac-MTADVDR- 2/3* One injection NH2 (SEQ ID NO: 5) per dayfor 9 200 μg/100 μl/injection constitutive days *When the two hind pawsof a mouse were inflamed, both paws were analyzed for swelling

TABLE 5A Experimental design (FIG. 5A) Group # Treatment #Mice Injectionprotocol 1 1XPBS 100 μl/injection 3 One injection per day for 9constitutive days 2 Peptide 5 mer Ac-MTADV- 5 One injection per day forNH2 (SEQ ID NO: 4) 9 constitutive days 200 μg/100 μl/injection 3Dexamethasone (Dex) 4 One injection per day for 9 constitutive days

TABLE 5B Experimental design (FIG. 5B) Group #Mice/ # Treatment Paws*Injection protocol 1 1XPBS 100 μ/injection 1 10/11* One injection perday for 10 constitutive days 2 Peptide 5 mer Ac-MTADV- 11/16* Oneinjection per day for NH2 (SEQ ID NO: 4) 10 constitutive days 200 μg/100μl/injection *When the two hind paws of a mouse were inflamed, both pawswere analyzed for swelling

TABLE 6 Experimental design (FIG. 6) Group # Treatment #Mice Injectionprotocol 1 1XPBS 100 μ/injection 1 13 One injection per day for 10constitutive days 2 Peptide 5 mer Ac-MTADV- 11 One injection per day NH2(SEQ ID NO: 4) for 10 constitutive days 70 μg/100 μl/injection

TABLE 7 Experimental design (FIGS. 7A-C) Group #Mice/ # Treatment Paws*Injection protocol 1 1XPBS 100 μl/injection 8/9* One injection per dayfor 9 constitutive days 2 Control: 7-mer scrambled 7/10* One injectionper day Peptide Ac-TMDVADR- for 9 constitutive days NH₂ (SEQ ID NO: 7)200 μg/100 μl/injection 3 Peptide 7-mer 9/11* One injection per dayAc-MTADVDR-NH₂ (SEQ ID for 9 constitutive days NO: 5) 200 μg/100μ/injection 1 *When the two hind paws of a mouse were inflamed, bothpaws were analyzed for swelling

Results

In the next step 9-7- and 5-mer RA peptides were synthesized withprotection residues, namely Acetyl and Amide residues at the amino andcarboxyl terminal ends of the peptides [Ac-TRMTADVDR-NH₂ (SEQ ID NO: 6),Ac-MTADVDR-NH₂ (SEQ ID NO: 5) and Ac-MTADV-NH₂ (SEQ ID NO: 4), denotedherein as 9-7- and 5-mer RA protected peptide, respectively]. Theseprotection residues preserve the natural stage of the peptide in theexperimental mouse and stabilize the peptide. The ability of theprotected peptides to reduce joint inflammation in DBA/1 mice followingtheir injection at the onset of CIA was evaluated. All measurements wereperformed under blind manner.

As can be seen in FIGS. 4 and 5A-B injection of all protected RApeptides at a dose of 200 μg per injection significantly inhibited jointinflammation in the CIA mice on DBA background, as compared to micetreated with PBS. As also evident in FIG. 5A, administration ofDexamethasone decreased footpad swelling as well, possibly by generatinga non-specific anti-inflammatory effect of this steroid. The 5-merprotected peptide (SEQ ID NO: 4) was also able to significantly inhibitjoint inflammation in the CIA mice on C57BL/6 background whenadministered at a dose of 70 μg per injection (FIG. 6). All measurementswere performed under blind manner.

In the next step, the effect of a scrambled RA peptide on jointinflammation was evaluated. To this end the effect of the 7-merprotected RA peptide (Ac-MTADVDR-NH₂, SEQ ID NO: 5) was compared to theeffect of a 7-mer scrambled protected peptide (Ac-TMDVADR-NH₂, SEQ IDNO: 7). As shown in FIGS. 7A-C, the scrambled non-specific 7-merprotected peptide had no effect on footpad swelling. All measurementswere performed under blind manner.

The results shown in FIGS. 7A-C were also evaluated by determining thepercent of healthy paws in each group. Hence, evaluation of the hindpaws of the CIA DBA/1 mice (FIG. 8), reflecting the severity of thedisease, showed that more than 60% of the hind paws in CIA mice treatedwith the 7-mer protected RA peptide remained healthy. In comparison, thepercentages of the hind paws that remained healthy in the other groupstested were 15% in the PBS treated group and 33% in the 7-mer peptidescrambled group.

Taken together, 5-, 7- and 9-mer RA protected peptides (SEQ ID NOs: 4-6)inhibit joint inflammation upon injection to CIA mice on both DBA andC57BL/6 background, while non-specific scrambled 7-mer protected peptide(SEQ ID NO: 7) has no effect on joint inflammation. Acetylation andAmidation did not affect the activity of the RA peptides (data notshown); however, they are expected to improve stability andpharmacokinetics of the RA peptide.

Example 4 70 μg Per Injection is the Optimal Dose for the 5-MerProtected RA Peptide for Reducing Joint Inflammation in the CIA MouseModel

Materials and Methods

Mice—

As described in Example 2 above.

Treatment Protocol—

PBS, or the tested peptide [Ac-MTADV-NH₂ (SEQ ID NO: 4)] at doses of 10,25, 70, 200 or 600 μg per injection were administered i.p to CIA mice 15for 10 consecutive days (see Tables 8-9 below). In all groups firstinjection was given at the onset of disease as determined by pawswelling.

Evaluation of Joint Inflammation—

As described in Example 2 above. The measurements were effected underblind manner.

Statistical Analysis—

As described in Example 2 above.

TABLE 8 Experimental design (FIG. 9A) Group # Treatment #Paws Injectionprotocol 1 1XPBS 100 μl/injection 13 One injection per day for 10constitutive days 2 5 mer Peptide Ac-MTADV- 11 One injection per day NH₂(SEQ ID NO: 4) for 10 constitutive days 70 μg/100 μl/injection 3 5 merPeptide Ac-MTADV- 11 One injection per day NH₂ (SEQ ID NO: 4) for 10constitutive days 200 μg/100 μl/injection 4 5 mer Peptide Ac-MTADV-  9One injection per day NH₂ (SEQ ID NO: 4) for 10 constitutive days 600μg/100 μl/injection

TABLE 9 Experimental design (FIG. 9B) Group # Treatment #Paws Injectionprotocol 1 1XPBS 100 μl/injection 13 One injection per day for 10constitutive days 2 5 mer Peptide Ac-MTADV- 11 One injection per day NH₂(SEQ ID NO: 4) for 10 constitutive days 10 μg/100 μl/injection 3 5 merPeptide Ac-MTADV- 11 One injection per day NH₂ (SEQ ID NO: 4) for 10constitutive days 25 μg/100 μl/injection 4 5 mer Peptide Ac-MTADV-  9One injection per day NH₂ (SEQ ID NO: 4) for 10 constitutive days 70μg/100 μl/injection

Results

To evaluate the optimal anti-inflammatory therapeutic dose of the 5-merRA peptide, several different doses of the protected peptide (SEQ ID NO:4) were administered to CIA mice on C57BL/6 background and jointinflammation was determined by footpad swelling measurements (electronicautomatic measurements of the volume of the footpad, based on Archimedesobservation). As can be seen in FIG. 9A, injection of 70, 200 or 600 μgper injection of the 5-mer protected RA peptide (SEQ ID NO: 4) reducedthe joint inflammation when compared to PBS control; however, a dose of70 μg of the peptide generated the most statistically significantanti-inflammatory effect. In addition, as shown 10 in FIG. 9B, injectionof 10 and 25 μg per injection of the 5-mer protected RA peptide (SEQ IDNO: 4) did not induce a significant anti-inflammatory effect.

Taken together, the data indicates that a dose of 70 μg per injection isthe optimal and the lowest dose for CIA inhibition by the 5-merprotected RA peptide (SEQ ID NO: 4).

Example 5 The 5-Mer RA Peptide does not Inhibit DTH in the CIA MouseModel

Materials and Methods

DTH Model—

C57BL/6 mice were painted at their abdomen with oxazolone 20 solution(sensitization). On day 6, the right ear of each mouse was painted withthe same hapten, oxazolone (elicitation), to generate delayed typehypersensitivity (DTH) response. The differences in thickness betweenthe right and the left ears, indicating DTH development, were determinedby microcaliper 24 hours later. DTH induction indicates a normal immuneresponse. For additional details see Weiss et al., (2000) Proc. Natl.Acad. Sci. USA. 97, 285-290.

Treatment Protocol—

PBS and 5-mer peptide (SEQ ID NO: 1) at a dose of 200 μg wasadministered one day before the sensitization and then every day duringthe sensitization period (7 days). An anti-TNF antibody (Herrring atal., (2002) Infect Immun 70, 2959-64) was used for comparison, todemonstrate non-specific effect.

Statistical Analysis—

As described in Example 2 above.

Results

To evaluate the influence the RA peptides on the immune response ingeneral the effect of the 5-mer RA peptide (SEQ ID NO: 1) on delayedtype hypersensitivity (DTH) response was evaluated in a DTH modelgenerated in C57BL/6 mice. DTH reflects acute inflammation,characterizing immune response against microorganism. The resultsindicate that injection of the 5-mer RA peptide did not affect the DTHresponse and mice treated with the peptide displayed the same DTHresponse as the control mice treated with PBS, throughout the 7 daysassay period (FIG. 10). In comparison mice treated with anti-TNFαantibody displayed inhibited DTH response, which was significantcompared to the control mice.

Example 6 The 5-Mer Peptide does not Generate Neutralizing Antibodies inthe CIA Mouse Model

Materials and Methods

Mice—

As described in Example 2 above.

Treatment Protocol—

PBS, or 5-mer RA peptide (SEQ ID NO: 1) at a dose of 70 μg per injectionwere administered i.p to CIA mice on C57BL/6 background for 10consecutive days. In all groups first injection was given at the onsetof disease as determined by paw swelling.

ELISA—

96 wells ELISA plates were coated with 5-mer RA peptide (SEQ ID NO: 1),collagen or immunoglobulin (positive control). Serum from CIA micetreated with PBS or the 5-mer RA peptide was added to the coated platesand the presence of neutralizing antibodies against collagen or the RApeptide was detected with anti-immunoglobulin+detection system. Theplate wells were coated with 1 mg/ml peptide or protein. Mouse serum wasadded to the plate wells for 15 hours in cold temperature. The detectionsystem included HRP-anti-mouse IgG and TMB (Bako) substrate. Plates wereanalysed using an ELISA reader at a wave length of 450 nm.

Statistical Analysis—

As described in Example 2 above.

Results

To determine whether treatment with the RA peptides induces productionof specific neutralizing antibodies that may reduce or even block thepeptide anti-inflammatory effect the abundance of anti-5-mer peptideantibodies in the serum was determined using an ELISA assay. As shown inFIG. 11, no neutralizing antibodies to the 5-mer RA peptide (SEQ IDNO: 1) were detected in the serum of CIA mice following treatment withthe peptide. Contrary, as the CIA mouse model is generated by collageninjection, anti-collagen specific antibodies were clearly evident in theserum of the CIA 5 mice.

Example 7 Serum Amyloid A, Transthyretin and Apolipoprotein B arePotential Target Proteins of the 5-Mer Peptide

Materials and Methods

Separation of Peptide Target Protein(s)—

Synovial fluid was removed from the joint of a Rheumatoid Arthritis (RA)patient. The synovial fluid was diluted 1:1 with PBS and centrifuged at1,200 rpm. The cell pellet was subjected to lysis buffer containingprotease inhibitors. The cell lysate was incubated with a biotinylated5-mer peptide (Sigma) or with PBS for 12 hour at 4° C., with shaking.Streptavidin Sepharose beads (Sephdex) were added to the biotinylated5-mer peptide-treated cell extract or PBS-treated cell extract foradditional one hour at 4° C., with shaking. The peptide-bound beads andthe control beads were separated by centrifugation, extensively washedand sent for mass spectrometry analysis, see FIG. 12. The massspectrometry (MS) measurements and analysis were performed in the SmolerProteomic Research Center at the Technion in Haifa.

Results

Mass spectrometry analysis of proteins from cell lysates extracted fromsynovial fluid cells of an RA patient that bound the 5-mer peptide (SEQID NO: 1) identified Serum Amyloid A (SAA), Transthyretin andApolipoprotein B as potential target proteins of the 5-mer RA peptide(SEQ ID NO: 1). The indicated proteins are known to be involved in thepathology of RA but also in the pathologies of Alzheimer's disease,cancer diseases and cardiovascular disease.

Example 8 Pharmacokinetics of the 5-Mer RA Peptide

Materials and Methods

Treatment Protocol—

C57BL/6 mice were subjected to a single i.p. injection of 200 μg 5-merpeptide (SEQ ID NO: 1). Blood samples were taken by terminal bleeding(500-1000 μl) 15, 35, and 60 minutes following injection and the serumwas sent for mass spectrometry evaluation.

Mass Spectrometry Analysis—

The concentration of the 5-mer RA peptide in the blood was determined bymass spectrum analysis as described above.

Results

The pharmacokinetic (PK) of the 5-mer RA peptide elimination in theblood of mice following a single i.p. injection is shown in FIG. 13.

Example 9 An In Vitro Model for Evaluating the Effect of the RA Peptides

Materials and Methods

Cells—

Fibroblasts from the inflammatory joint of an RA patient were culturedand maintained as shown in Bendersky et al, J Immunol.; 188:4349-59,2012. A quantity of 20,000 cells was added to each one of the 96 wellplates with the indicated concentrations of peptide, serum amyloid A orα-lactalbumin.

MTT Assay—

MTT assay was effected as shown in Madhyastha et al. (2015) J Clin DiagnRes.; 9:ZC05-8.

Statistical Analysis—

As described in Example 2 above.

Results

An in-vitro model was developed as a tool to evaluate the biologicalactivity of the generated RA peptides. To this end, fibroblasts from theinflammatory joint of an RA patient were incubated in-vitro and theeffect of the peptides on cell survival was evaluated by a MTT assay.

As shown in FIG. 14, increasing the dose of the 5-mer RA peptide (SEQ IDNO: 1) gradually inhibits cell survival. Addition 50 μg/ml Serum AmyloidA (SAA) into the fibroblast culture in combination with the 5-merpeptide prevents this inhibition. The results also indicate that a lowdose of 2.5 μg/ml (˜5 nM) of the peptide is able to significantlyinhibit cell survival in this in-vitro model, and that the in-vitromaximal suppressive effect is 60%.

In order to demonstrate the specificity of the effect of the peptide oncell survival, the peptide was added to the cell culture at a constantconcentration (25 μg/ml) and SAA or lactalbumin (LA, a control proteinwith similar molecular weight) was added at excalating concentrations.As shown in FIG. 15, the 5-mer peptide reduced survival of thefibroblast and addition of SAA gradually prevented this reduction in adose response manner. In contrast, the addition of LA had no effect onthe suppressive activity the peptide; indicating the SAA prevents theinhibitory effect of the 5-mer peptide in a specific manner.

In the next step, the suppressive effect of the 5-mer protected RApeptide (SEQ ID NO: 4) was compared to the 5-mer RA peptide (SEQ ID NO:1). As shown in FIG. 16, peptide modification improved the suppressiveeffect of the peptide.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. An isolated polypeptide consisting of an amino acid sequence selectedform the group consisting of SEQ ID NOs: 1-3.
 2. An isolated end-cappingmodified polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-3, wherein said modified polypeptidecomprises an anti-inflammatory activity.
 3. The end-capping modifiedpolypeptide of claim 2, wherein said end-capping comprises an N terminusend-capping.
 4. The end-capping modified polypeptide of claim 3, whereinsaid N terminus end-capping comprises an Acetyl.
 5. The end-cappingmodified polypeptide of claim 2, wherein said end-capping comprises a Cterminus end-capping.
 6. The end-capping modified polypeptide of claim5, wherein said C terminus end-capping comprises an Amide.
 7. Theend-capping modified polypeptide of claim 2, wherein said polypeptideconsists of an amino acid sequence selected form the group consisting ofSEQ ID NOs: 1-3.
 8. The isolated peptide of claim 1, wherein saidpolypeptide is as set forth in SEQ ID NO:
 1. 9. The end-capping modifiedpolypeptide of claim 2, being selected from the group consisting to SEQID NOs: 4-6.
 10. A composition of matter comprising the isolatedpolypeptide of claim 1 and a non-proteinaceous moiety attached to saidisolated polypeptide, wherein said isolated fusion polypeptide comprisesan anti-inflammatory activity.
 11. An isolated fusion polypeptidecomprising the isolated polypeptide of claim 1 having a C and/or Nterminally attached amino acid sequence, wherein said C terminally aminoacid sequence is a non-contiguous CD44vRA amino acid sequence with saidisolated fusion polypeptide; and wherein said fusion polypeptidecomprises an anti-inflammatory activity.
 12. The composition of matterof claim 10, wherein said attached is covalent attachment.
 13. Theisolated polypeptide of claim 2, wherein said anti-inflammatory activityis not dependent on vaccination or mucosal tolerance.
 14. The isolatedpolypeptide of claim 2, being capable of binding a protein selected fromthe group consisting of serum amyloid A, Transthyretin andapolipoprotein B.
 15. A pharmaceutical composition comprising as anactive agent the isolated polypeptide of claim 1; and a pharmaceuticallyacceptable carrier or diluent.
 16. A method of treating an inflammatorydisease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of theisolated polypeptide of claim 1, thereby treating the inflammatorydisease in the subject. 17-19. (canceled)
 20. The method of claim 16,wherein said inflammatory disease involves cells expressing CD44vRA. 21.The method of claim 16, wherein said inflammatory disease is selectedfrom the group consisting of Rheumatoid arthritis, multiple sclerosis,psoriatic arthritis, Alzheimer's disease, cancer and cardiovasculardisease.
 22. The method of claim 16, wherein said inflammatory diseaseis multiple sclerosis.
 23. An isolated polynucleotide comprising anucleic acid sequence encoding the isolated polypeptide of claim 1.24-29. (canceled)